Glucagon/GLP-1 receptor co-agonists

ABSTRACT

Provided herein are peptides and variant peptides that exhibit enhanced activity at the GLP-1 receptor, as compared to native glucagon.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/494,484, filed on Jun. 12, 2012, which is now U.S. Pat. No. 9,156,902and claims priority to U.S. Provisional Application No. 61/500,027,filed Jun. 22, 2011, and U.S. Provisional Application No. 61/547,360,filed Oct. 14, 2011, each of which are incorporated by reference intheir entirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 12 kilobytes ACII (Text) file named“240262_SeqList.txt,” created on Sep. 4, 2015.

BACKGROUND

Pre-proglucagon is a 158 amino acid precursor polypeptide that isprocessed in different tissues to form a number of differentproglucagon-derived peptides, including glucagon, glucagon-likepeptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin(OXM), that are involved in a wide variety of physiological functions,including glucose homeostasis, insulin secretion, gastric emptying, andintestinal growth, as well as the regulation of food intake. Glucagon isa 29-amino acid peptide that corresponds to amino acids 33 through 61 ofpre-proglucagon, while GLP-1 is produced as a 37-amino acid peptide thatcorresponds to amino acids 72 through 108 of pre-proglucagon.GLP-1(7-36) amide or GLP-1(7-37) acid are biologically potent forms ofGLP-1, that demonstrate essentially equivalent activity at the GLP-1receptor.

During hypoglycemia, when blood glucose levels drop below normal,glucagon signals the liver to break down glycogen and release glucose,causing blood glucose levels to rise toward a normal level. Hypoglycemiais a common side effect of insulin therapy in patients withhyperglycemia (elevated blood glucose levels) due to diabetes. Thus,glucagon's most recognized role in glucose regulation is to counteractthe action of insulin and maintain blood glucose levels.

GLP-1 has different biological activities compared to glucagon. Itsactions include stimulation of insulin synthesis and secretion,inhibition of glucagon secretion, and inhibition of food intake. GLP-1has been shown to reduce hyperglycemia in diabetics. Exendin-4, apeptide from lizard venom that shares about 50% amino acid identity withGLP-1, activates the GLP-1 receptor and likewise has been shown toreduce hyperglycemia in diabetics.

There is also evidence that GLP-1 and exendin-4 may reduce food intakeand promote weight loss, an effect that would be beneficial not only fordiabetics but also for patients suffering from obesity. Patients withobesity have a higher risk of diabetes, hypertension, hyperlipidemia,cardiovascular disease, and musculoskeletal diseases.

SUMMARY

The present disclosures provide peptides and variant peptides thatexhibit activity at the glucagon receptor, activity at the GLP-1receptor, or activity at each of the glucagon receptor and the GLP-1receptor. In exemplary embodiments, the presently disclosed peptides andvariant peptides exhibit enhanced activity at the GLP-1 receptor, ascompared to native glucagon. In exemplary aspects, the peptides andvariant peptides exhibit at least 100-fold selectivity for the humanGLP-1 receptor versus the GIP receptor.

The present disclosures further provide conjugates comprising any of thepeptides and variant peptides described herein conjugated to aheterologous moiety. In exemplary aspects, the heterologous moiety is apeptide or protein and the conjugate is a fusion peptide or chimericpeptide. In exemplary aspects, the heterologous moiety is a polymer,e.g., a polyethylene glycol. The present disclosures furthermore providedimers and multimers comprising any of the peptides and variant peptidesdescribed herein.

The present disclosures moreover provides pharmaceutical compositionscomprising any of the peptides and variant peptides described herein anda pharmaceutically acceptable carrier, as well as a method of treatingor preventing a disease or medical condition (e.g., metabolic syndrome,diabetes, obesity, liver steatosis, a neurodegenerative disease,hypoglycemia) in a patient. The method comprises administering to thepatient a presently disclosed peptide or peptide variant, optionallyformulated into a pharmaceutical composition, in an amount effective totreat the disease or medical condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a graph of the cumulative body weight change (grams)of DIO mice treated with a vehicle control or a dose of a peptide of SEQID NO: 12, 17, 18, or 19, as detailed in Example 7.

FIG. 2 represents a graph of the basal glucose (mg/dl) of DIO micetreated with a vehicle control or a dose of a peptide of SEQ ID NO: 12,17, 18, or 19, as detailed in Example 7.

FIG. 3A represents a graph of the cumulative body weight change (%) ofobese rhesus monkeys treated with a vehicle control, Liraglutide (Lira),or a dose of a peptide of SEQ ID NO: 17 or 20, as detailed in Example 8.

FIG. 3B represents a graph of the cumulative food intake (expressed as apercent of food intake on Day 0) of obese rhesus monkeys treated with avehicle control, Liraglutide, or a dose of a peptide of SEQ ID NO: 17 or20, as detailed in Example 8.

FIG. 4 represents a graph of blood glucose levels (mg/dL) of diabeticrhesus monkeys treated with a vehicle control or a dose of a peptide ofSEQ ID NO: 17, as detailed in Example 9.

DETAILED DESCRIPTION Definitions

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating diabetes” willrefer in general to altering glucose blood levels in the direction ofnormal levels and may include increasing or decreasing blood glucoselevels depending on a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a glucagon peptide refers to a nontoxic but sufficient amountof the peptide to provide the desired effect. For example one desiredeffect would be the prevention or treatment of hyperglycemia, e.g., asmeasured by a change in blood glucose level closer to normal, orinducing weight loss/preventing weight gain, e.g., as measured byreduction in body weight, or preventing or reducing an increase in bodyweight, or normalizing body fat distribution. The amount that is“effective” will vary from subject to subject, depending on the age andgeneral condition of the individual, mode of administration, and thelike. Thus, it is not always possible to specify an exact “effectiveamount.” However, an appropriate “effective” amount in any individualcase may be determined by one of ordinary skill in the art using routineexperimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route, e.g., subcutaneous, intramuscular, intraspinal, orintravenous.

As used herein, the term “peptide” encompasses a chain of 3 or moreamino acids and typically less than 100 amino acids, wherein the aminoacids are naturally occurring or coded or non-naturally occurring ornon-coded amino acids. Non-naturally occurring amino acids refer toamino acids that do not naturally occur in vivo but which, nevertheless,can be incorporated into the peptide structures described herein.“Non-coded” as used herein refers to an amino acid that is not anL-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val,Trp, Tyr. “Coded” as used herein refers to an amino acid that is anL-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val,Trp, Tyr. In some embodiments, the peptides and variant peptidesdescribed herein are about the same length as SEQ ID NO: 1 (which is 29amino acids in length), e.g. 25-35 amino acids in length. Exemplarylengths include 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

Typically, polypeptides and proteins have a polymer length that isgreater than that of “peptides.”

Throughout the application, all references to a particular amino acidposition by number (e.g., position 28) refer to the amino acid at thatposition in native glucagon (SEQ ID NO: 1) or the corresponding aminoacid position in any analogs thereof. For example, a reference herein to“position 28” would mean the corresponding position 27 for a glucagonanalog in which the first amino acid of SEQ ID NO: 1 has been deleted.Similarly, a reference herein to “position 28” would mean thecorresponding position 29 for a glucagon analog in which one amino acidhas been added before the N-terminus of SEQ ID NO: 1. As used herein an“amino acid modification” refers to (i) a substitution or replacement ofan amino acid of SEQ ID NO: 1 with a different amino acid(naturally-occurring or coded or non-coded or non-naturally-occurringamino acid), (ii) an addition of an amino acid (naturally-occurring orcoded or non-coded or non-naturally-occurring amino acid), to SEQ ID NO:1 or (iii) a deletion of one or more amino acids of SEQ ID NO: 1.

“Percent identity” with respect to two amino acid sequences refers tothe number of amino acids of the first sequence that match (areidentical to) the amino acids in the second reference sequence, dividedby the length of the reference sequence, when the two sequences arealigned to achieve maximum correspondence (e.g. gaps can be introducedfor optimal alignment).

Amino acid “modification” refers to an insertion, deletion orsubstitution of one amino acid with another. In some embodiments, theamino acid substitution or replacement is a conservative amino acidsubstitution, e.g., a conservative substitution of the amino acid at oneor more of positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20,21, 24, 27, 28 or 29. As used herein, the term “conservative amino acidsubstitution” is the replacement of one amino acid with another aminoacid having similar properties, e.g., size, charge, hydrophobicity,hydrophilicity, and/or aromaticity, and includes exchanges within one ofthe following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negative-charged residues and their amides and esters:

-   -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;

III. Polar, positive-charged residues:

-   -   His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

-   -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp, acetyl phenylalanine

In some embodiments, the amino acid substitution is not a conservativeamino acid substitution, e.g., is a non-conservative amino acidsubstitution.

As used herein the term “charged amino acid” or “charged residue” refersto an amino acid that comprises a side chain that is negative-charged(i.e., de-protonated) or positive-charged (i.e., protonated) in aqueoussolution at physiological pH. For example negative-charged amino acidsinclude aspartic acid, glutamic acid, cysteic acid, homocysteic acid,and homoglutamic acid, whereas positive-charged amino acids includearginine, lysine and histidine. Charged amino acids include the chargedamino acids among the 20 coded amino acids, as well as atypical ornon-naturally occurring or non-coded amino acids.

As used herein the term “acidic amino acid” refers to an amino acid thatcomprises a second acidic moiety (other than the carboxylic acid of theamino acid), including for example, a carboxylic acid or sulfonic acidgroup.

As used herein, the term “acylated amino acid” refers to an amino acidcomprising an acyl group which is non-native to a naturally-occurringamino acid, regardless of the means by which it is produced (e.g.acylation prior to incorporating the amino acid into a peptide, oracylation after incorporation into a peptide).

As used herein the term “alkylated amino acid” refers to an amino acidcomprising an alkyl group which is non-native to a naturally-occurringamino acid, regardless of the means by which it is produced.Accordingly, the acylated amino acids and alkylated amino acids of thepresent disclosures are non-coded amino acids.

As used herein, the term “selectivity” of a molecule for a firstreceptor relative to a second receptor refers to the following ratio:EC50 of the molecule at the second receptor divided by the EC50 of themolecule at the first receptor. For example, a molecule that has an EC50of 1 nM at a first receptor and an EC50 of 100 nM at a second receptorhas 100-fold selectivity for the first receptor relative to the secondreceptor.

As used herein the term “native glucagon” refers to a peptide consistingof the sequence of SEQ ID NO: 1 and the term “native GLP-1” is a genericterm that designates GLP-1(7-36) amide, GLP-1(7-37) acid or a mixture ofthose two compounds.

As used herein, “glucagon potency” or “potency compared to nativeglucagon” of a molecule refers to the inverse ratio of the EC50 of themolecule at the glucagon receptor divided by the EC50 of native glucagonat glucagon receptor.

As used herein, “GLP-1 potency” or “potency compared to native GLP-1” ofa molecule refers to the inverse ratio of the EC50 of the molecule atGLP-1 receptor divided by the EC50 of native GLP-1 at GLP-1 receptor.

EMBODIMENTS

The present disclosures provide peptides and variant peptides thatexhibit activity at the GLP-1 receptor, at the glucagon receptor, or atboth the GLP-1 receptor and the glucagon receptor. In this regard, thepresent disclosures provide GLP-1 receptor agonist peptides, glucagonreceptor agonist peptides, and GLP-1/glucagon receptor co-agonistpeptides. In exemplary embodiments, the presently disclosed peptides andvariant peptides exhibit enhanced activity or greater potency at theGLP-1 receptor, as compared to native human glucagon (SEQ ID NO: 1). Inexemplary embodiments, the peptides and variant peptides of the presentdisclosures exhibit greater potency at the GLP-1 receptor as compared tonative human GLP-1 (SEQ ID NO: 2) or one of the active forms thereof(SEQ ID NOs: 5 and 6). In exemplary embodiments, the peptides andvariant peptides exhibit greater potency at the glucagon receptorcompared to native human GLP-1. In exemplary embodiments, the peptidesand variant peptides exhibit greater potency at the glucagon receptorcompared to native human glucagon.

In exemplary embodiments, the peptides and variant peptides describedherein exhibit other improvements in properties relative to nativeglucagon or native GLP-1, such as greater stability, greater solubility,a prolonged half-life in circulation, a delayed onset of action, anextended duration of action, a dampened peak (e.g., relatively decreasedmean peak plasma concentration), and an improved resistance toproteases, such as DPP-W.

The peptides and variant peptides described herein are based on theamino acid sequence of native human glucagon (SEQ ID NO: 1), and aredescribed herein as “peptides”, “variant peptides”, “glucagon analogs”,“analogs”, or “glucagon peptides.” It is understood that terms such as“analog” or “variant” or “modifications” encompass peptides or proteinssynthesized de novo and do not require the performance of any particularmodification step. In some aspects, the peptides and variant peptidesdescribed herein comprise a modified amino acid sequence of SEQ ID NO: 1comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acidmodifications relative to SEQ ID NO: 1, and in some instances, 16 ormore (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, 26), amino acidmodifications, as further described herein. The following description ofglucagon analogs and/or glucagon peptides thus applies to any of thepresently disclosed peptides and variant peptides, regardless of thedegree of similarity between native human glucagon (SEQ ID NO: 1) andthe peptide or variant peptide of the present disclosures.

It is contemplated that any of the peptide sequences described hereinmay be further varied by incorporating additional amino acidmodifications; for example, by including any of the modificationsdescribed herein, e.g., at the positions described herein, or byincorporating conservative substitutions, or by returning to the nativeglucagon amino acid (see SEQ ID NO: 1) at that position. In exemplaryembodiments, the modifications include, e.g., acylation, alkylation,pegylation, truncation at C-terminus, substitution of the amino acid atone or more of positions 1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21,23, 24, 27, 28, and 29. For example, where any of the peptide sequencesdisclosed herein includes a Cys for purposes of pegylation, a variantpeptide may use a different amino acid for pegylation. As anotherexample, a variant peptide may be pegylated at a different position(e.g., replacing the existing Cys with a different amino acid, insertinga new Cys at the proposed pegylation position, and pegylating the newCys). As yet a further example, where any of the peptide sequencesdisclosed herein includes a Lys for purposes of acylation, the Lys maybe moved to a different position and the new position acylated. In anyof the embodiments described herein, the variant peptides may be, forexample, 80%, 85%, 90% or 95% identical to the parent peptides over thelength of the parent peptides or over amino acids 1-29 of the parentpeptide (e.g., may incorporate 1, 2, 3, 4, or 5 additional modificationscompared to the parent peptide).

Conjugates, fusion proteins and multimers of any of the peptidesequences disclosed herein are also contemplated.

Activity of the Peptides and Variant Peptides

Agonist Activity at the Glucagon Receptor

In exemplary embodiments, the peptides and variant peptides of thepresent disclosures exhibit an EC50 at the glucagon receptor of about1000 μM or less (e.g., about 750 μM or less, about 500 μM or less, about250 μM or less, about 100 μM or less, about 75 μM or less, about 50 μMor less, about 25 μM or less, about 10 μM or less, about 5 μM or less,or about 1 μM or less). In exemplary embodiments, the peptides andvariant peptides exhibit an EC50 for glucagon receptor activation whichis in the nanomolar range. For example, the presently disclosed peptidesand variant peptides exhibit an EC50 at the glucagon receptor which isabout 1000 nM or less (e.g., about 750 nM or less, about 500 nM or less,about 250 nM or less, about 100 nM or less, about 75 nM or less, about50 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM orless, or about 1 nM or less). In exemplary embodiments, the peptides andvariant peptides exhibit an EC50 at the glucagon receptor which is inthe picomolar range. Accordingly, in exemplary aspects, the peptides andvariant peptides exhibit an EC50 for glucagon receptor activation ofabout 1000 pM or less (e.g., about 750 pM or less, about 500 pM or less,about 250 pM or less, about 100 pM or less, about 75 pM or less, about50 pM or less, about 25 pM or less, about 10 pM or less, about 5 pM orless, or about 1 pM or less). It is understood that a lower EC50indicates higher activity or potency at the receptor.

In some embodiments, the glucagon analogs described herein exhibit anEC50 at the glucagon receptor that is about 0.001 pM or more, about 0.01pM or more, or about 0.1 pM or more. Glucagon receptor activation(glucagon receptor activity) can be measured by in vitro assaysmeasuring cAMP induction in HEK293 cells over-expressing the glucagonreceptor, e.g., assaying HEK293 cells co-transfected with DNA encodingthe glucagon receptor and a luciferase gene linked to cAMP responsiveelement as described in Example 2.

In exemplary embodiments, the presently disclosed peptides and variantpeptides exhibit about 0.001% or more, about 0.01% or more, about 0.1%or more, about 0.5% or more, about 1% or more, about 5% or more, about10% or more, about 20% or more, about 30% or more, about 40% or more,about 50% or more, about 60% or more, about 75% or more, about 100% ormore, about 125% or more, about 150% or more, about 175% or more, about200% or more, about 250% or more, about 300% or more, about 350% ormore, about 400% or more, about 450% or more, or about 500% or higheractivity at the glucagon receptor relative to native glucagon (glucagonpotency). In some embodiments, the peptides and variant peptidesdescribed herein exhibit about 5000% or less or about 10,000% or lessactivity at the glucagon receptor relative to native glucagon. Aglucagon analog's activity at a receptor relative to a native ligand ofthe receptor is calculated as the inverse ratio of EC50s for theglucagon analog vs. the native ligand.

In some embodiments, the peptides and variant peptides exhibitsubstantial activity (potency) at only the glucagon receptor and littleto no activity at the GLP-1 receptor. Accordingly, in some embodiments,the peptides and variant peptides are considered as “pure glucagonreceptor agonists” or are not considered as a “glucagon/GLP-1 receptorco-agonist.” In some embodiments these peptides and variant peptidesexhibit any of the levels of activity or potency at the glucagonreceptor described herein but have substantially less activity (potency)at the GLP-1 receptor. In some embodiments, the glucagon analog exhibitsan EC50 at the GLP-1 receptor which is 100-fold or greater than the EC50at the glucagon receptor.

Agonist Activity at the GLP-1 Receptor

In exemplary embodiments, the peptides and variant peptides exhibit anEC50 for GLP-1 receptor activation of about 1000 μM or less (e.g., about750 μM or less, about 500 μM or less, about 250 μM or less, about 100 μMor less, about 75 μM or less, about 50 μM or less, about 25 μM or less,about 10 μM or less, about 5 μM or less, or about 1 μM or less). Inexemplary embodiments, the peptides and variant peptides exhibit an EC50at the GLP-1 receptor of about 1000 nM or less (e.g., about 750 nM orless, about 500 nM or less, about 250 nM or less, about 100 nM or less,about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10nM or less, about 5 nM or less, or about 1 nM or less). In exemplaryembodiments, the peptides and variant peptides has an EC50 at the GLP-1receptor which is in the picomolar range. Accordingly, in someembodiments, the peptides and variant peptides exhibit an EC50 for GLP-1receptor activation of about 1000 pM or less (e.g., about 750 pM orless, about 500 pM or less, about 250 pM or less, about 100 pM or less,about 75 pM or less, about 50 pM or less, about 25 pM or less, about 10pM or less, about 5 pM or less, or about 1 pM or less). It is understoodthat a lower EC50 indicates higher activity or potency at the receptor.

In exemplary embodiments, the peptides and variant peptides describedherein exhibit an EC50 at the GLP-1 receptor that is about 0.001 pM ormore, about 0.01 pM or more, or about 0.1 pM or more. GLP-1 receptoractivation (GLP-1 receptor activity) can be measured by in vitro assaysmeasuring cAMP induction in HEK293 cells over-expressing the GLP-1receptor, e.g., assaying HEK293 cells co-transfected with DNA encodingthe GLP-1 receptor and a luciferase gene linked to cAMP responsiveelement as described in Example 2.

In some embodiments, the peptides and variant peptides of the presentdisclosures exhibit about 0.001% or more, about 0.01% or more, about0.1% or more, about 0.5% or more, about 1% or more, about 5% or more,about 10% or more, about 20% or more, about 30% or more, about 40% ormore, about 50% or more, about 60% or more, about 75% or more, about100% or more, about 125% or more, about 150% or more, about 175% ormore, about 200% or more, about 250% or more, about 300% or more, about350% or more, about 400% or more, about 450% or more, or about 500% orhigher activity at the GLP-1 receptor relative to native GLP-1 (GLP-1potency). In some embodiments, the peptides and variant peptidesdescribed herein exhibit about 5000% or less or about 10,000% or lessactivity at the GLP-1 receptor relative to native GLP-1 (GLP-1 potency).

In some embodiments, the peptides and variant peptides exhibitsubstantial activity (potency) at only the GLP-1 receptor and little tono activity at the glucagon receptor. In some embodiments, the peptidesand variant peptides are considered as “pure GLP-1 receptor agonists” orare not considered as “glucagon/GLP-1 receptor co-agonists.” In someembodiments these peptides and variant peptides exhibit any of thelevels of activity or potency at the GLP-1 receptor described herein buthave substantially less activity (potency) at the glucagon receptor. Insome embodiments, the peptides and variant peptides exhibit an EC50 atthe glucagon receptor which is 100-fold or greater than the EC50 at theGLP-1 receptor.

Agonist Activity at the GLP-1 Receptor and the Glucagon Receptor

In exemplary embodiments, the peptides and variant peptides exhibitactivity at both the GLP-1 receptor and glucagon receptor and may beconsidered as “glucagon/GLP-1 receptor co-agonists”. In exemplaryembodiments, the activity (e.g., the EC50 or the relative activity orpotency) of the peptides and variant peptides at the glucagon receptoris within about 50-fold, about 40-fold, about 30-fold, about 20-fold,about 10-fold, or about 5 fold different (higher or lower) from itsactivity (e.g., the EC50 or the relative activity or potency) at theGLP-1 receptor. In exemplary aspects, the glucagon potency of thepeptide or variant peptide is within about 25-, about 20-, about 15-,about 10-, or about 5-fold different (higher or lower) from its GLP-1potency. In exemplary aspects, the glucagon potency of the peptide orvariant peptide is within about 25-, about 20-, about 15-, about 10-, orabout 5-fold lower from its GLP-1 potency.

In exemplary embodiments, the co-agonist is approximately equipotent orrelatively more potent at the GLP-1 receptor than the glucagon receptor.For example, the ratio of the relative activity or the EC50 or thepotency of the peptide or variant peptide at the glucagon receptordivided by the relative activity or the EC50 or potency of the peptideor variant peptide at the GLP-1 receptor is less than, or is about, X,wherein X is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. Inexemplary embodiments, the ratio of the EC50 or potency or relativeactivity of the peptide or variant peptide at the glucagon receptordivided by the EC50 or potency or relative activity of the peptide orvariant peptide at the GLP-1 receptor is about 1 and less than 5 (e.g.,about 4, about 3, about 2, about 1). In exemplary embodiments, the ratioof the EC50 or potency or relative activity of the peptide or variantpeptide at the GLP-1 receptor divided by the EC50 or potency or relativeactivity of the peptide or variant peptide at the glucagon receptor isless than 5 (e.g., about 4, about 3, about 2, about 1). In exemplaryembodiments, the ratio of the glucagon potency of the peptide or variantpeptide compared to the GLP-1 potency of the peptide or variant peptideis less than, or is about, Y, wherein Y is selected from 100, 75, 60,50, 40, 30, 20, 15, 10, and 5. In exemplary embodiments, the ratio ofthe glucagon potency of the peptide or variant peptide compared to theGLP-1 potency of the peptide or variant peptide is less than 5 (e.g.,about 4, about 3, about 2, about 1). In some embodiments, the glucagonanalog has an EC50 at the glucagon receptor which is 2- to 10-fold(e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold) greater than the EC50 at the GLP-1 receptor.

In exemplary embodiments, the peptide is primarily a glucagon agonistand is relatively more potent at the glucagon receptor than the GLP-1receptor (e.g. the peptide is 5 times or more potent at the glucagonreceptor compared to the GLP-1 receptor). For example, the ratio of therelative activity or potency or the EC50 of the peptide or variantpeptide at the GLP-1 receptor divided by the relative activity orpotency or the EC50 of the peptide or variant peptide at the glucagonreceptor is less than, or is about, V, wherein V is selected from 100,75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio ofthe GLP-1 potency of the peptide or variant peptide compared to theglucagon potency of the peptide or variant peptide is less than, or isabout, W, wherein W is selected from 100, 75, 60, 50, 40, 30, 20, 15,10, and 5. In some embodiments, the peptide or variant peptide exhibitsat least 0.1% (e.g., about 0.5% or more, about 1% or more, about 5% ormore, about 10% or more, or more) of the activity of native GLP-1 at theGLP-1 receptor (GLP-1 potency) and exhibits at least 0.1% (e.g., about0.5% or more, about 1% or more, about 5% or more, about 10% or more, ormore) of the activity of native glucagon at the glucagon receptor(glucagon potency).

Activity at the GIP Receptor

In addition to being active at the glucagon receptor and/or the GLP-1receptor, the peptides and variant peptides described herein, in someaspects, exhibit low agonist activity at the GIP receptor. In suchaspects, preferably such peptides and variant peptides are at least100-fold selective for the GLP-1 receptor relative to the GIP receptor.

In other aspects, however, the peptide or variant peptide exhibitsappreciable activity at the GIP receptor, e.g. the EC50 of the analog atthe GIP receptor is less than about 50-fold different from its EC50 atthe GLP-1 receptor, optionally, wherein the GIP potency of the analog iswithin about 50-fold of the GLP-1 potency of the analog. In exemplaryembodiments, the peptides exhibit an EC50 for GIP receptor activationactivity of about 1 μM or less, or 100 nM or less, or about 75, 50, 25,10, 8, 6, 5, 4, 3, 2 or 1 nM or less. It is understood that a lower EC50indicates higher activity or potency at the receptor. In someembodiments, the peptides and variant peptides described herein exhibitan EC50 at the GIP receptor that is about 0.001 nM, 0.01 nM, or 0.1 nM.In some embodiments, the peptides and variant peptides described hereinexhibit an EC50 at the GIP receptor that is no more than about 100 nM.Receptor activation can be measured by in vitro assays measuring cAMPinduction in HEK293 cells over-expressing the receptor, e.g. assayingHEK293 cells co-transfected with DNA encoding the receptor and aluciferase gene linked to cAMP responsive element as described inExample 2.

In some embodiments, the presently disclosed peptides and variantpeptides exhibit at least about 0.1%, 1%, 10%, 50%, 100%, 150%, or 200%or higher activity at the GIP receptor relative to native GIP (GIPpotency). In some embodiments, the peptides and variant peptidesdescribed herein exhibit no more than 1000%, 10,000%, 100,000%, or1,000,000% activity at the GIP receptor relative to native GIP. Aglucagon peptide's activity (potency) at a receptor relative to a nativeligand of the receptor is calculated as the inverse ratio of EC50s forthe peptide vs. the native ligand.

Thus, one aspect of the present disclosures provides peptides andvariant peptides that exhibit activity at both the glucagon receptor andthe GIP receptor (“glucagon/GIP co-agonists”). In some embodiments, theEC50 of the peptide at the GIP receptor is less than about 50-fold,40-fold, 30-fold or 20-fold different (higher or lower) from its EC50 atthe glucagon receptor. In some embodiments, the GIP potency of thepeptide is less than about 500-, 450-, 400-, 350-, 300-, 250-, 200-,150-, 100-, 75-, 50-, 25-, 20-, 15-, 10-, or 5-fold different (higher orlower) from its glucagon potency. In some embodiments, GLP-1 activityhas been significantly reduced or destroyed, e.g., by an amino acidmodification at position 7, a deletion of the amino acid(s) C-terminalto the amino acid at position 27 or 28, or a combination thereof.

In alternative aspects of the present disclosures, the peptides andvariant peptides of the present disclosures exhibit activity at theGLP-1 and GIP receptors, but do not exhibit significant activity at theglucagon receptor (“GIP/GLP-1 co-agonists”), e.g., due to an amino acidmodification of Gln at position 3. For example, substitution at thisposition with an acidic, basic, or a hydrophobic amino acid (glutamicacid, ornithine, norleucine) reduces glucagon activity. In otheraspects, the peptides and variant peptides exhibit activity at each ofthe glucagon, GIP and GLP-1 receptors (“glucagon/GIP/GLP-1tri-agonists”). For example, in either of these latter aspects, the EC50of the peptide at the GIP receptor is less than about 50-fold, 40-fold,30-fold or 20-fold different (higher or lower) from its EC50 at theGLP-1 receptor. In some embodiments, the GIP potency of the peptide isless than about 25-, 20-, 15-, 10-, or 5-fold different (higher orlower) from its GLP-1 potency. In some embodiments these peptides haveabout 10% or less of the activity of native glucagon at the glucagonreceptor, e.g. about 1-10%, or about 0.1-10%, or greater than about 0.1%but less than about 10%.

Activity of Conjugates

In some embodiments, the peptides and variant peptides described hereinexhibit activity or potency at the glucagon receptor and/or activity atthe GLP-1 receptor and/or activity at the GIP receptor, as describedabove and, when the peptide or variant peptide is part of a conjugate(e.g., is conjugated to a heterologous moiety, e.g., a hydrophilicmoiety, e.g., a polyethylene glycol), the peptide or variant peptideexhibits an activity that is lower (i.e. lower potency or higher EC50)than when the peptide or variant peptide is not part of the conjugate.In some aspects, the peptide or variant peptide when not part ofconjugate exhibits a potency at the glucagon receptor and/or the GLP-1receptor that is about 10-fold or greater than the potency of thepeptide or variant peptide when part of a conjugate. In some aspects,the peptide or variant peptide when unconjugated exhibits an potency atthe glucagon receptor and/or GLP-1 receptor that is about 10-fold, about15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold,about 40-fold, about 45-fold, about 50-fold, about 100-fold, or evengreater-fold the potency of the peptide or variant peptide whenconjugated.

Structure of the Glucagon Analogs

Acylation

In accordance with some embodiments, the glucagon analog comprises anacylated amino acid (e.g., a non-coded acylated amino acid (e.g., anamino acid comprising an acyl group which is non-native to anaturally-occurring amino acid)). The acylated amino acid in someembodiments causes the glucagon analog to have one or more of (i) aprolonged half-life in circulation, (ii) a delayed onset of action,(iii) an extended duration of action, (iv) an improved resistance toproteases, such as DPP-IV, and (v) increased potency at one or both ofthe GLP-1 and glucagon receptors. As shown herein, acylated glucagonanalogs do not exhibit decreased activity at the glucagon and GLP-1receptors in comparison to the corresponding unacylated glucagon analog.Rather, in some instances, acylated glucagon analogs actually exhibitincreased activity at the GLP-1 and glucagon receptors. Accordingly, thepotency of the acylated glucagon analogs is comparable to the unacylatedversions of the glucagon analogs, if not enhanced.

In accordance with one embodiment, the glucagon analog comprises an acylgroup which is attached to the glucagon analog via an ester, thioester,or amide linkage for purposes of prolonging half-life in circulationand/or delaying the onset of and/or extending the duration of actionand/or improving resistance to proteases such as DPP-IV.

Acylation can be carried out at any position within the glucagon analog,including any of positions 1-29, a position C-terminal to the 29^(th)amino acid (e.g., the amino acid at position 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, etc., at a position within aC-terminal extension or at the C-terminus), provided that glucagonand/or GLP-1 activity is retained, if not enhanced. Nonlimiting examplesinclude positions 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24,27, 28, or 29. In exemplary embodiments, the glucagon analog comprisesan acylated amino acid at one or more positions selected from the groupconsisting of: 9, 10, 12, 16, and 20. In exemplary embodiments, theglucagon analog comprises an acylated amino acid at one or morepositions selected from the group consisting of: 10, 12, and 16. Inexemplary embodiments, the glucagon analog comprises an acylated aminoacid at one or more positions selected from the group consisting of: 9,10, 12, 16, and 20. In exemplary embodiments, the glucagon analogcomprises an acylated amino acid at one or more positions 10 and 12. Inexemplary embodiments, the glucagon analog comprises an acylated aminoacid at position 12. In exemplary embodiments, the glucagon analogcomprises a C-terminal extension and an acylated amino acid at one ormore positions selected from the group consisting of 9, 10, 12, 16, 20,and 37-43 (e.g., 40). In specific embodiments, acylation occurs atposition 10 of the glucagon analog and the glucagon analog lacks anintramolecular bridge, e.g., a covalent intramolecular bridge (e.g., alactam bridge). Such acylated glucagon analogs lacking an intramolecularbridge demonstrate enhanced activity at the GLP-1 and glucagon receptorsas compared to the corresponding non-acylated analogs lacking a covalentintramolecular bridge and in comparison to the corresponding analogslacking an intramolecular bridge acylated at a position other thanposition 10. As shown herein, acylation at position 10 can eventransform a glucagon analog having little activity at the glucagonreceptor to a glucagon analog having activity at both the glucagon andGLP-1 receptors. Accordingly, the position at which acylation occurs canalter the overall activity profile of the glucagon analog.

The glucagon analog in some embodiments are acylated at the same aminoacid position where a hydrophilic moiety is linked, or at a differentamino acid position. Nonlimiting examples include acylation at position10 and pegylation at one or more positions in the C-terminal portion ofthe glucagon analog, e.g., position 24, 28 or 29, within a C-terminalextension, or at the C-terminus (e.g., through adding a C-terminal Cys).

The acyl group can be covalently linked directly to an amino acid of theglucagon analog, or indirectly to an amino acid of the glucagon analogvia a spacer, wherein the spacer is positioned between the amino acid ofthe glucagon analog and the acyl group.

In specific aspects, the glucagon analog is modified to comprise an acylgroup by direct acylation of an amine, hydroxyl, or thiol of a sidechain of an amino acid of the glucagon analog. In some embodiments,acylation is at position 10, 20, 24, or 29 of the glucagon analog. Inthis regard, the acylated glucagon analog can comprise the amino acidsequence of SEQ ID NO: 1, or a modified amino acid sequence thereofcomprising one or more of the amino acid modifications described herein,with at least one of the amino acids at positions 10, 20, 24, and 29 ofthe analog modified to any amino acid comprising a side chain amine,hydroxyl, or thiol. In some specific embodiments, the direct acylationof the glucagon analog occurs through the side chain amine, hydroxyl, orthiol of the amino acid at position 10.

In some embodiments, the amino acid comprising a side chain amine is anamino acid of Formula I:

In some exemplary embodiments, the amino acid of Formula I, is the aminoacid wherein n is 4 (Lys) or n is 3 (Orn).

In other embodiments, the amino acid comprising a side chain hydroxyl isan amino acid of Formula II:

In some exemplary embodiments, the amino acid of Formula II is the aminoacid wherein n is 1 (Ser).

In yet other embodiments, the amino acid comprising a side chain thiolis an amino acid of Formula III:

In some exemplary embodiments, the amino acid of Formula III is theamino acid wherein n is 1 (Cys).

In yet other embodiments, the amino acid comprising a side chain amine,hydroxyl, or thiol is a disubstituted amino acid comprising the samestructure of Formula I, Formula II, or Formula III, except that thehydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

In some embodiments, the acylated glucagon comprises a spacer betweenthe analog and the acyl group. In some embodiments, the glucagon analogis covalently bound to the spacer, which is covalently bound to the acylgroup.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol. The aminoacid to which the spacer is attached can be any amino acid (e.g., asingly or doubly α-substituted amino acid) comprising a moiety whichpermits linkage to the spacer. For example, an amino acid comprising aside chain NH₂, —OH, or —COOH (e.g., Lys, Orn, Ser, Asp, or Glu) issuitable. In this respect, the acylated glucagon analog can comprise theamino acid sequence of SEQ ID NO: 1, or a modified amino acid sequencethereof comprising one or more of the amino acid modifications describedherein, with at least one of the amino acids at positions 10, 20, 24,and 29 modified to any amino acid comprising a side chain amine,hydroxyl, or carboxylate.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol.

When acylation occurs through an amine group of a spacer, the acylationcan occur through the alpha amine of the amino acid or a side chainamine. In the instance in which the alpha amine is acylated, the aminoacid of the spacer can be any amino acid. For example, the amino acid ofthe spacer can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu,Ile, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid,7-aminoheptanoic acid, and 8-aminooctanoic acid. Alternatively, theamino acid of the spacer can be an acidic residue, e.g., Asp, Glu,homoglutamic acid, homocysteic acid, cysteic acid, gamma-glutamic acid.

In the instance in which the side chain amine of the amino acid of thespacer is acylated, the amino acid of the spacer is an amino acidcomprising a side chain amine, e.g., an amino acid of Formula I (e.g.,Lys or Orn). In this instance, it is possible for both the alpha amineand the side chain amine of the amino acid of the spacer to be acylated,such that the glucagon analog is diacylated. Embodiments of theinvention include such diacylated molecules.

When acylation occurs through a hydroxyl group of a spacer, the aminoacid or one of the amino acids of the dipeptide or tripeptide can be anamino acid of Formula II. In a specific exemplary embodiment, the aminoacid is Ser.

When acylation occurs through a thiol group of a spacer, the amino acidor one of the amino acids of the dipeptide or tripeptide can be an aminoacid of Formula III. In a specific exemplary embodiment, the amino acidis Cys.

In some embodiments, the spacer is a hydrophilic bifunctional spacer. Incertain embodiments, the hydrophilic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophilic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophilic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophilic bifunctional spacer comprises a thiol group and acarboxylate. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In some embodiments, the spacer is a hydrophobic bifunctional spacer.Hydrophobic bifunctional spacers are known in the art. See, e.g.,Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego,Calif., 1996), which is incorporated by reference in its entirety. Incertain embodiments, the hydrophobic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophobic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophobic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophobic bifunctional spacer comprises a thiol group and acarboxylate. Suitable hydrophobic bifunctional spacers comprising acarboxylate and a hydroxyl group or a thiol group are known in the artand include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoicacid.

In some embodiments, the bifunctional spacer is not a dicarboxylic acidcomprising an unbranched, methylene of 1-7 carbon atoms between thecarboxylate groups. In some embodiments, the bifunctional spacer is adicarboxylic acid comprising an unbranched, methylene of 1-7 carbonatoms between the carboxylate groups.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional spacer, or hydrophobic bifunctional spacer) in specificembodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or10 atoms) in length. In more specific embodiments, the spacer is about 3to 10 atoms (e.g., 6 to 10 atoms) in length and the acyl group is a C12to C18 fatty acyl group, e.g., C14 fatty acyl group, C16 fatty acylgroup, such that the total length of the spacer and acyl group is 14 to28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, or 28 atoms. In some embodiments, the length of the spacer andacyl group is 17 to 28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with certain foregoing embodiments, the bifunctionalspacer can be a synthetic or naturally occurring amino acid (including,but not limited to, any of those described herein) comprising an aminoacid backbone that is 3 to 10 atoms in length (e.g., 6-amino hexanoicacid, 5-aminovaleric acid, 7-aminoheptanoic acid, and 8-aminooctanoicacid). Alternatively, the spacer can be a dipeptide or tripeptide spacerhaving a peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) inlength. Each amino acid of the dipeptide or tripeptide spacer can be thesame as or different from the other amino acid(s) of the dipeptide ortripeptide and can be independently selected from the group consistingof: naturally-occurring or coded and/or non-coded or non-naturallyoccurring amino acids, including, for example, any of the D or L isomersof the naturally-occurring amino acids (Ala, Cys, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr), or anyD or L isomers of the non-naturally occurring or non-coded amino acidsselected from the group consisting of: β-alanine (β-Ala),N-α-methyl-alanine (Me-Ala), aminobutyric acid (Abu), γ-aminobutyricacid (γ-Abu), aminohexanoic acid (ε-Ahx), aminoisobutyric acid (Aib),aminomethylpyrrole carboxylic acid, aminopiperidinecarboxylic acid,aminoserine (Ams), aminotetrahydropyran-4-carboxylic acid, arginineN-methoxy-N-methyl amide, β-aspartic acid (β-Asp), azetidine carboxylicacid, 3-(2-benzothiazolyl)alanine, α-tert-butylglycine,2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine(Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab),diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),dimethylthiazolidine (DMTA), γ-Glutamic acid (γ-Glu), homoserine (Hse),hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide,methyl-isoleucine (MeIle), isonipecotic acid (Isn), methyl-leucine(MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone(Met(O₂)), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline(Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine(Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO₂)),4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg),piperidinylalanine, piperidinylglycine, 3,4-dehydroproline,pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec),O-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA),1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic),tetrahydropyranglycine, thienylalanine (Thi), O-benzyl-phosphotyrosine,O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine,O-(bis-dimethylamino-phosphono)-tyrosine, tyrosine sulfatetetrabutylamine, methyl-valine (MeVal), and alkylated3-mercaptopropionic acid.

In some embodiments, the spacer comprises an overall negative charge,e.g., comprises one or two negative-charged amino acids. In someembodiments, the dipeptide is not any of the dipeptides of generalstructure A-B, wherein A is selected from the group consisting of Gly,Gln, Ala, Arg, Asp, Asn, Ile, Leu, Val, Phe, and Pro, wherein B isselected from the group consisting of Lys, His, Trp. In someembodiments, the dipeptide spacer is selected from the group consistingof: Ala-Ala, β-Ala-β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyricacid-γ-aminobutyric acid, Glu-Glu, and γ-Glu-γ-Glu.

In some exemplary embodiments, the glucagon analog is modified tocomprise an acyl group by acylation of an amine, hydroxyl, or thiol of aspacer, which spacer is attached to a side chain of an amino acid atposition 10, 20, 24, or 29, or at the C-terminal amino acid of theglucagon analog.

In yet more specific embodiments, the acyl group is attached to theamino acid at position 10 of the glucagon analog and the length of thespacer and acyl group is 14 to 28 atoms. The amino acid at position 10,in some aspects, is an amino acid of Formula I, e.g., Lys, or adisubstituted amino acid related to Formula I. In more specificembodiments, the glucagon analog lacks an intramolecular bridge, e.g., acovalent intramolecular bridge. The glucagon analog, for example, can bea glucagon analog comprising one or more alpha, alpha-disubstitutedamino acids, e.g., AIB, for stabilizing the alpha helix of the analog.

Suitable methods of peptide acylation via amines, hydroxyls, and thiolsare known in the art. See, for example, Example 19 (for methods ofacylating through an amine), Miller, Biochem Biophys Res Commun 218:377-382 (1996); Shimohigashi and Stammer, Int J Pept Protein Res 19:54-62 (1982); and Previero et al., Biochim Biophys Acta 263: 7-13 (1972)(for methods of acylating through a hydroxyl); and San and Silvius, JPept Res 66: 169-180 (2005) (for methods of acylating through a thiol);Bioconjugate Chem. “Chemical Modifications of Proteins: History andApplications” pages 1, 2-12 (1990); Hashimoto et al., PharmacueticalRes. “Synthesis of Palmitoyl Derivatives of Insulin and their BiologicalActivity” Vol. 6, No: 2 pp. 171-176 (1989).

The acyl group of the acylated amino acid can be of any size, e.g., anylength carbon chain, and can be linear or branched. In some specificembodiments, the acyl group is a C4 to C30 fatty acid. For example, theacyl group can be any of a C4 fatty acid, C6 fatty acid, C8 fatty acid,C10 fatty acid, C12 fatty acid, C14 fatty acid, C16 fatty acid, C18fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid, C26 fattyacid, C28 fatty acid, or a C30 fatty acid. In some embodiments, the acylgroup is a C8 to C20 fatty acid, e.g., a C14 fatty acid or a C16 fattyacid.

In an alternative embodiment, the acyl group is a bile acid. The bileacid can be any suitable bile acid, including, but not limited to,cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid,taurocholic acid, glycocholic acid, and cholesterol acid.

In some embodiments, the glucagon analog comprises an acylated aminoacid by acylation of a long chain alkane by the glucagon analog. Inspecific aspects, the long chain alkane comprises an amine, hydroxyl, orthiol group (e.g., octadecylamine, tetradecanol, and hexadecanethiol)which reacts with a carboxyl group, or activated form thereof, of theglucagon analog. The carboxyl group, or activated form thereof, of theglucagon analog can be part of a side chain of an amino acid (e.g.,glutamic acid, aspartic acid) of the glucagon analog or can be part ofthe analog backbone.

In certain embodiments, the glucagon analog is modified to comprise anacyl group by acylation of the long chain alkane by a spacer which isattached to the glucagon analog. In specific aspects, the long chainalkane comprises an amine, hydroxyl, or thiol group which reacts with acarboxyl group, or activated form thereof, of the spacer. Suitablespacers comprising a carboxyl group, or activated form thereof, aredescribed herein and include, for example, bifunctional spacers, e.g.,amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacersand hydrophobic bifunctional spacers.

As used herein, the term “activated form of a carboxyl group” refers toa carboxyl group with the general formula R(C═O)X, wherein X is aleaving group and R is the glucagon analog or the spacer. For example,activated forms of a carboxyl groups may include, but are not limitedto, acyl chlorides, anhydrides, and esters. In some embodiments, theactivated carboxyl group is an ester with a N-hydroxysuccinimide ester(NHS) leaving group.

With regard to these aspects, in which a long chain alkane is acylatedby the glucagon analog or the spacer, the long chain alkane may be ofany size and can comprise any length of carbon chain. The long chainalkane can be linear or branched. In certain aspects, the long chainalkane is a C4 to C30 alkane. For example, the long chain alkane can beany of a C4 alkane, C6 alkane, C8 alkane, C10 alkane, C12 alkane, C14alkane, C16 alkane, C18 alkane, C20 alkane, C22 alkane, C24 alkane, C26alkane, C28 alkane, or a C30 alkane. In some embodiments, the long chainalkane comprises a C8 to C20 alkane, e.g., a C14 alkane, C16 alkane, ora C18 alkane.

Also, in some embodiments, an amine, hydroxyl, or thiol group of theglucagon analog is acylated with a cholesterol acid. In a specificembodiment, the glucagon analog is linked to the cholesterol acidthrough an alkylated des-amino Cys spacer, i.e., an alkylated3-mercaptopropionic acid spacer. The alkylated des-amino Cys spacer canbe, for example, a des-amino-Cys spacer comprising a dodecaethyleneglycol moiety. In one embodiment, the glucagon analog comprises thestructure:

The acylated glucagon analogs described herein can be further modifiedto comprise a hydrophilic moiety. In some specific embodiments thehydrophilic moiety can comprise a polyethylene glycol (PEG) chain. Theincorporation of a hydrophilic moiety can be accomplished through anysuitable means, such as any of the methods described herein. In thisregard, the acylated glucagon analog can comprise SEQ ID NO: 1,including any of the modifications described herein, in which at leastone of the amino acids at position 10, 20, 24, and 29 of the analogcomprises an acyl group and at least one of the amino acids at position16, 17, 21, 24, or 29, a position within a C-terminal extension, or theC-terminal amino acid are modified to a Cys, Lys, Orn, homo-Cys, orAc-Phe, and the side chain of the amino acid is covalently bonded to ahydrophilic moiety (e.g., PEG). In some embodiments, the acyl group isattached to position 10, optionally via a spacer comprising Cys, Lys,Orn, homo-Cys, or Ac-Phe, and the hydrophilic moiety is incorporated ata Cys residue at position 24.

Alternatively, the acylated glucagon analog can comprise a spacer,wherein the spacer is both acylated and modified to comprise thehydrophilic moiety. Nonlimiting examples of suitable spacers include aspacer comprising one or more amino acids selected from the groupconsisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

Alkylation

In accordance with some embodiments, the glucagon analog comprises analkylated amino acid (e.g., a non-coded alkylated amino acid (e.g., anamino acid comprising an alkyl group which is non-native to anaturally-occurring amino acid)). Without being held to any particulartheory, it is believed that alkylation of glucagon analogs achievesimilar, if not the same, effects as acylation of the glucagon analogs,e.g., a prolonged half-life in circulation, a delayed onset of action,an extended duration of action, an improved resistance to proteases,such as DPP-IV, and increased potency at the GLP-1 and glucagonreceptors.

Alkylation can be carried out at any positions within the glucagonanalog, including any of the positions described herein as a site foracylation, including but not limited to, any of amino acid positions1-29, an amino acid position C-terminal to the 29^(th) residue, e.g.,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,etc., at a position within a C-terminal extension, or at the C-terminus,provided that the glucagon activity or GLP-1 activity is retained.Nonlimiting examples include positions 5, 7, 10, 11, 12, 13, 14, 16, 17,18, 19, 20, 21, 24, 27, 28, or 29. In exemplary embodiments, theglucagon analog comprises an alkylated amino acid at one or morepositions selected from the group consisting of: 9, 10, 12, 16, and 20.In exemplary embodiments, the glucagon analog comprises an alkylatedamino acid at one or more positions selected from the group consistingof: 10, 12, and 16. In exemplary embodiments, the glucagon analogcomprises an alkylated amino acid at one or more positions selected fromthe group consisting of: 9, 10, 12, 16, and 20. In exemplaryembodiments, the glucagon analog comprises an alkylated amino acid atone or more positions 10 and 12. In exemplary embodiments, the glucagonanalog comprises an alkylated amino acid at position 12. In exemplaryembodiments, the glucagon analog comprises a C-terminal extension and analkylated amino acid at one or more positions selected from the groupconsisting of 9, 10, 12, 16, 20, and 37-43 (e.g., 40). The alkyl groupcan be covalently linked directly to an amino acid of the glucagonanalog, or indirectly to an amino acid of the glucagon analog via aspacer, wherein the spacer is positioned between the amino acid of theglucagon analog and the alkyl group. Glucagon analog may be alkylated atthe same amino acid position where a hydrophilic moiety is linked, or ata different amino acid position. Nonlimiting examples include alkylationat position 10 and pegylation at one or more positions in the C-terminalportion of the glucagon analog, e.g., position 24, 28 or 29, within aC-terminal extension, or at the C-terminus (e.g., through adding aC-terminal Cys).

In specific aspects, the glucagon analog is modified to comprise analkyl group by direct alkylation of an amine, hydroxyl, or thiol of aside chain of an amino acid of the glucagon analog. In some embodiments,alkylation is at position 10, 20, 24, or 29 of the glucagon analog. Inthis regard, the alkylated glucagon analog can comprise the amino acidsequence of SEQ ID NO: 2, or a modified amino acid sequence thereofcomprising one or more of the amino acid modifications described herein,with at least one of the amino acids at positions 10, 20, 24, and 29modified to any amino acid comprising a side chain amine, hydroxyl, orthiol. In some specific embodiments, the direct alkylation of theglucagon analog occurs through the side chain amine, hydroxyl, or thiolof the amino acid at position 10.

In some embodiments, the amino acid comprising a side chain amine is anamino acid of Formula I. In some exemplary embodiments, the amino acidof Formula I, is the amino acid wherein n is 4 (Lys) or n is 3 (Orn).

In other embodiments, the amino acid comprising a side chain hydroxyl isan amino acid of Formula II. In some exemplary embodiments, the aminoacid of Formula II is the amino acid wherein n is 1 (Ser).

In yet other embodiments, the amino acid comprising a side chain thiolis an amino acid of Formula III. In some exemplary embodiments, theamino acid of Formula III is the amino acid wherein n is 1 (Cys).

In yet other embodiments, the amino acid comprising a side chain amine,hydroxyl, or thiol is a disubstituted amino acid comprising the samestructure of Formula I, Formula II, or Formula III, except that thehydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

In some embodiments, the alkylated glucagon analog comprises a spacerbetween the analog and the alkyl group. In some embodiments, theglucagon analog is covalently bound to the spacer, which is covalentlybound to the alkyl group. In some exemplary embodiments, the glucagonanalog is modified to comprise an alkyl group by alkylation of an amine,hydroxyl, or thiol of a spacer, which spacer is attached to a side chainof an amino acid at position 10, 20, 24, or 29 of the glucagon analog.The amino acid to which the spacer is attached can be any amino acidcomprising a moiety which permits linkage to the spacer. For example, anamino acid comprising a side chain NH₂, —OH, or —COOH (e.g., Lys, Orn,Ser, Asp, or Glu) is suitable. In this respect, the alkylated glucagonanalog can comprise a modified amino acid sequence of SEQ ID NO: 1,comprising one or more of the amino acid modifications described herein,with at least one of the amino acids at positions 10, 20, 24, and 29modified to any amino acid comprising a side chain amine, hydroxyl, orcarboxylate.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol.

When alkylation occurs through an amine group of a spacer, thealkylation can occur through the alpha amine of an amino acid or a sidechain amine. In the instance in which the alpha amine is alkylated, theamino acid of the spacer can be any amino acid. For example, the aminoacid of the spacer can be a hydrophobic amino acid, e.g., Gly, Ala, Val,Leu, Ile, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovalericacid, 7-aminoheptanoic acid, and 8-aminooctanoic acid. Alternatively,the amino acid of the spacer can be an acidic residue, e.g., Asp andGlu, provided that the alkylation occurs on the alpha amine of theacidic residue. In the instance in which the side chain amine of theamino acid of the spacer is alkylated, the amino acid of the spacer isan amino acid comprising a side chain amine, e.g., an amino acid ofFormula I (e.g., Lys or Orn). In this instance, it is possible for boththe alpha amine and the side chain amine of the amino acid of the spacerto be alkylated, such that the glucagon analog is dialkylated.Embodiments of the invention include such dialkylated molecules.

When alkylation occurs through a hydroxyl group of a spacer, the aminoacid or one of the amino acids of the dipeptide or tripeptide can be anamino acid of Formula II. In a specific exemplary embodiment, the aminoacid is Ser.

When alkylation occurs through a thiol group of spacer, the amino acidor one of the amino acids of the dipeptide or tripeptide can be an aminoacid of Formula III. In a specific exemplary embodiment, the amino acidis Cys.

In some embodiments, the spacer is a hydrophilic bifunctional spacer. Incertain embodiments, the hydrophilic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophilic bifunctional spacer is comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophilic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophilic bifunctional spacer comprises a thiol group and acarboxylate. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In some embodiments, the spacer is a hydrophobic bifunctional spacer. Incertain embodiments, the hydrophobic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophobic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydropholic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydropholic bifunctional spacer comprises a thiol group and acarboxylate. Suitable hydrophobic bifunctional spacers comprising acarboxylate and a hydroxyl group or a thiol group are known in the artand include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoicacid.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional spacer, or hydrophobic bifunctional spacer) in specificembodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or10 atoms)) in length. In more specific embodiments, the spacer is about3 to 10 atoms (e.g., 6 to 10 atoms) in length and the alkyl is a C12 toC18 alkyl group, e.g., C14 alkyl group, C16 alkyl group, such that thetotal length of the spacer and alkyl group is 14 to 28 atoms, e.g.,about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28atoms. In some embodiments, the length of the spacer and alkyl is 17 to28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with certain foregoing embodiments, the bifunctionalspacer can be a synthetic or non-naturally occurring or non-coded aminoacid comprising an amino acid backbone that is 3 to 10 atoms in length(e.g., 6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoicacid, and 8-aminooctanoic acid). Alternatively, the spacer can be adipeptide or tripeptide spacer having a peptide backbone that is 3 to 10atoms (e.g., 6 to 10 atoms) in length. The dipeptide or tripeptidespacer can be composed of naturally-occurring or coded and/or non-codedor non-naturally occurring amino acids, including, for example, any ofthe amino acids taught herein. In some embodiments, the spacer comprisesan overall negative charge, e.g., comprises one or two negative-chargedamino acids. In some embodiments, the dipeptide spacer is selected fromthe group consisting of: Ala-Ala, β-Ala-β-Ala, Leu-Leu, Pro-Pro,γ-aminobutyric acid-γ-aminobutyric acid, and γ-Glu-γ-Glu.

Suitable methods of peptide alkylation via amines, hydroxyls, and thiolsare known in the art. For example, a Williamson ether synthesis can beused to form an ether linkage between a hydroxyl group of the glucagonanalog and the alkyl group. Also, a nucleophilic substitution reactionof the peptide with an alkyl halide can result in any of an ether,thioether, or amino linkage.

The alkyl group of the alkylated glucagon analog can be of any size,e.g., any length carbon chain, and can be linear or branched. In someembodiments, the alkyl group is a C4 to C30 alkyl. For example, thealkyl group can be any of a C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl,C26 alkyl, C28 alkyl, or a C30 alkyl. In some embodiments, the alkylgroup is a C8 to C20 alkyl, e.g., a C14 alkyl or a C16 alkyl.

In some specific embodiments, the alkyl group comprises a steroid moietyof a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholicacid, lithocholic acid, taurocholic acid, glycocholic acid, andcholesterol acid.

In some embodiments of the disclosure, the glucagon analog comprises analkylated amino acid by reacting a nucleophilic, long chain alkane withthe glucagon analog, wherein the glucagon analog comprises a leavinggroup suitable for nucleophilic substitution. In specific aspects, thenucleophilic group of the long chain alkane comprises an amine,hydroxyl, or thiol group (e.g., octadecylamine, tetradecanol, andhexadecanethiol). The leaving group of the glucagon analog can be partof a side chain of an amino acid or can be part of the peptide backbone.Suitable leaving groups include, for example, N-hydroxysuccinimide,halogens, and sulfonate esters.

In certain embodiments, the glucagon analog is modified to comprise analkyl group by reacting the nucleophilic, long chain alkane with aspacer which is attached to the glucagon analog, wherein the spacercomprises the leaving group. In specific aspects, the long chain alkanecomprises an amine, hydroxyl, or thiol group. In certain embodiments,the spacer comprising the leaving group can be any spacer discussedherein, e.g., amino acids, dipeptides, tripeptides, hydrophilicbifunctional spacers and hydrophobic bifunctional spacers furthercomprising a suitable leaving group.

With regard to these aspects of the disclosure, in which a long chainalkane is alkylated by the glucagon analog or the spacer, the long chainalkane may be of any size and can comprise any length of carbon chain.The long chain alkane can be linear or branched. In certain aspects, thelong chain alkane is a C4 to C30 alkane. For example, the long chainalkane can be any of a C4 alkane, C6 alkane, C8 alkane, C10 alkane, C12alkane, C14 alkane, C16 alkane, C18 alkane, C20 alkane, C22 alkane, C24alkane, C26 alkane, C28 alkane, or a C30 alkane. In some embodiments,the long chain alkane comprises a C8 to C20 alkane, e.g., a C14 alkane,C16 alkane, or a C18 alkane.

Also, in some embodiments, alkylation can occur between the glucagonanalog and a cholesterol moiety. For example, the hydroxyl group ofcholesterol can displace a leaving group on the long chain alkane toform a cholesterol-glucagon analog product.

The alkylated glucagon analogs described herein can be further modifiedto comprise a hydrophilic moiety. In some specific embodiments thehydrophilic moiety can comprise a polyethylene glycol (PEG) chain. Theincorporation of a hydrophilic moiety can be accomplished through anysuitable means, such as any of the methods described herein. In thisregard, the alkylated glucagon analog can comprise a modified SEQ ID NO:1 comprising one or more of the amino acid modifications describedherein, in which at least one of the amino acids at position 10, 20, 24,and 29 comprise an alkyl group and at least one of the amino acids atposition 16, 17, 21, 24, and 29, a position within a C-terminalextension or the C-terminal amino acid are modified to a Cys, Lys, Orn,homo-Cys, or Ac-Phe, and the side chain of the amino acid is covalentlybonded to a hydrophilic moiety (e.g., PEG). In some embodiments, thealkyl group is attached to position 10, optionally via a spacercomprising Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the hydrophilicmoiety is incorporated at a Cys residue at position 24.

Alternatively, the alkylated glucagon analog can comprise a spacer,wherein the spacer is both alkylated and modified to comprise thehydrophilic moiety. Nonlimiting examples of suitable spacers include aspacer comprising one or more amino acids selected from the groupconsisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

Stabilization of the Alpha Helix and Alpha Helix Promoting Amino Acids

Without being bound to any particular theory, the glucagon analogsdescribed herein comprise a helical structure, e.g., an alpha helix. Insome embodiments, the glucagon analog comprises amino acids whichstabilize the alpha helical structure. Accordingly, in some aspects, theglucagon analog comprises one or more alpha helix promoting amino acids.As used herein, the term “alpha helix promoting amino acid” refers to anamino acid which provides increased stability to an alpha helix of theglucagon analog of which it is a part. Alpha helix promoting amino acidsare known in the art. See, for example, Lyu et al., Proc Natl Acad SciU.S.A. 88: 5317-5320 (1991); Branden & Tooze, Introduction to ProteinStructure, Garland Publishing, New York, N.Y., 1991; Fasman, Predictionof Protein Structure and the Principles of Protein Conformation, ed.Fasman, Plenum, N Y, 1989). Suitable alpha helix promoting amino acidsfor purposes herein include, but are not limited to: alanine, norvaline,norleucine, alpha aminobutyric acid, alpha-aminoisobutyric acid,leucine, isoleucine, valine, and the like. In some embodiments, thealpha helix promoting amino acid is any amino acid which is part of analpha helix found in a naturally-occurring protein, e.g., Leu, Phe, Ala,Met, Gly, Ile, Ser, Asn, Glu, Asp, Lys, Arg.

In some embodiments, the alpha helix promoting amino acid provides morestability to the alpha helix as compared to glycine or alanine. In someembodiments, the alpha helix promoting amino acid is an alpha, alphadi-substituted amino acid.

Alpha Helix: Position of Alpha Helix Promoting Amino Acids

In some embodiments, the glucagon analog comprises an amino acidsequence which is similar to native glucagon (SEQ ID NO: 1) and theglucagon analog comprises at least one alpha helix promoting amino acid.In some embodiments, the alpha helix promoting amino acid is located atany of positions 12 to 29 (according to the numbering of native glucagon(SEQ ID NO: 1). In some embodiments, the glucagon analog comprises amodified amino acid sequence of SEQ ID NO: 1 and comprises at least onealpha helix promoting amino acid, e.g., at one or more of positions 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. Insome embodiments, the glucagon analog comprises an alpha helix promotingamino acid at one, two, three, or all of positions 16, 17, 20, and 21.

Alpha Helix: Alpha, Alpha Di-Substituted Amino Acids

In some embodiments, the alpha helix promoting amino acid is analpha,alpha di-substituted amino acid. In specific embodiments, thealpha, alpha di-substituted amino acid comprises R¹ and R², each ofwhich is bonded to the alpha carbon, wherein each of R¹ and R² isindependently selected from the group consisting of C1-C4 alkyl,optionally substituted with a hydroxyl, amide, thiol, halo, or R¹ and R²together with the alpha carbon to which they are attached form a ring(e.g., a C3-C8 ring). In some embodiments, each of R¹ and R² is selectedfrom the group consisting of: methyl, ethyl, propyl, and n-butyl, or R¹and R² together form a cyclooctane or cycloheptane (e.g.,1-aminocyclooctane-1-carboxylic acid). In some embodiments, R¹ and R²are the same. In some embodiments, R¹ is different from R². In certainaspects, each of R¹ and R² is a C1-C4 alkyl. In some aspects, each of R¹and R² is a C1 or C2 alkyl. In some embodiments, each of R¹ and R² ismethyl, such that the alpha, alpha disubstituted amino acid isalpha-aminoisobutyric acid (AIB).

In some aspects, the glucagon analogs described herein comprises one ormore alpha, alpha di-substituted amino acids and the glucagon analogsspecifically lack a covalent intramolecular bridge (e.g., a lactam),since the alpha, alpha disubstituted amino acid is capable ofstabilizing the alpha helix in the absence of a covalent bridge. In someaspects, the glucagon analog comprises one or more alpha, alphadi-substituted amino acids at the C-terminus (around positions 12-29).In some embodiments, one, two, three, four or more of positions 16, 17,18, 19, 20, 21, 24, 28, or 29 of the glucagon analog is substituted withan α, α-disubstituted amino acid, e.g., amino iso-butyric acid (AIB), anamino acid disubstituted with the same or a different group selectedfrom methyl, ethyl, propyl, and n-butyl, or with a cyclooctane orcycloheptane (e.g., 1-aminocyclooctane-1-carboxylic acid). For example,substitution of position 16 with AIB enhances GLP-1 activity, in theabsence of an intramolecular bridge, e.g., a non-covalent intramolecularbridge (e.g., a salt bridge) or a covalent intramolecular bridge (e.g.,a lactam). In some embodiments, one, two, three or more of positions 16,20, 21 or 24 are substituted with AIB. In specific embodiments, one orboth of the amino acids corresponding to positions 2, 16, of nativehuman glucagon (SEQ ID NO: 1) are substituted with an alpha, alphadisubstituted amino acid such as AIB.

In accordance with some embodiments, the glucagon analog lacking anintramolecular bridge comprises one or more substitutions within aminoacid positions 12-29 with an α,α-disubstituted amino acid and an acyl oralkyl group covalently attached to the side chain of the amino acid atposition 10 of the glucagon analog. In specific embodiments, the acyl oralkyl group is not naturally occurring on an amino acid. In certainaspects, the acyl or alkyl group is non-native to the amino acid atposition 10. Such acylated or alkylated glucagon peptides lacking anintramolecular bridge exhibit enhanced activity at the GLP-1 andglucagon receptors as compared to the non-acylated counterpart peptides.Further enhancement in activity at the GLP-1 and glucagon receptors canbe achieved by the acylated glucagon peptides lacking an intramolecularbridge by incorporating a spacer between the acyl or alkyl group and theside chain of the amino acid at position 10 of the analog. Acylation andalkylation, with or without incorporating spacers, are further describedherein.

Alpha Helix: Intramolecular Bridges

In some embodiments, the alpha helix promoting amino acid is an aminoacid which is linked to another amino acid of the glucagon analog via anintramolecular bridge. In such embodiments, each of these two aminoacids linked via an intramolecular bridge is considered an alpha helixpromoting amino acid. In some embodiments, the glucagon analog comprisesone or two intramolecular bridges. In some specific embodiments, theglucagon analog comprises one intramolecular bridge in combination withat least one other alpha helix promoting amino acid, e.g., an alpha,alpha-disubstituted amino acid.

In some embodiments, the intramolecular bridge is a bridge whichconnects two parts of the glucagon analog via noncovalent bonds,including, for example, van der Waals interactions, hydrogen bonds,ionic bonds, hydrophobic interactions, dipole-dipole interactions, andthe like. In this regard, the glucagon analog in certain aspectscomprises a non-covalent intramolecular bridge. In some embodiments, thenon-covalent intramolecular bridge is a salt bridge.

In some embodiments, the intramolecular bridge is a bridge whichconnects two parts of the analog via covalent bonds. In this regard, theglucagon analog in certain aspects comprises a covalent intramolecularbridge.

In some embodiments, the intramolecular bridge (e.g., non-covalentintramolecular bridge, covalent intramolecular bridge) is formed betweentwo amino acids that are 3 amino acids apart, e.g., amino acids atpositions i and i+4, wherein i is any integer between 12 and 25 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25). Moreparticularly, the side chains of the amino acid pairs 12 and 16, 16 and20, 20 and 24 or 24 and 28 (amino acid pairs in which i=12, 16, 20, or24) are linked to one another and thus stabilize the glucagon alphahelix. Alternatively, i can be 17. In some specific embodiments, theglucagon analog comprises an intramolecular bridge between amino acids17 and 21. In some specific embodiments, the glucagon analog comprisesan intramolecular bridge between the amino acids at positions 16 and 20or 12 and 16 and a second intramolecular bridge between the amino acidsat positions 17 and 21. Glucagon analogs comprising one or moreintramolecular bridges are contemplated herein. In specific embodiments,wherein the amino acids at positions i and i+4 are joined by anintramolecular bridge, the size of the linker is about 8 atoms, or about7-9 atoms.

In other embodiments, the intramolecular bridge is formed between twoamino acids that are two amino acids apart, e.g., amino acids atpositions j and j+3, wherein j is any integer between 12 and 26 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26). In somespecific embodiments, j is 17. In specific embodiments, wherein aminoacids at positions j and j+3 are joined by an intramolecular bridge, thesize of the linker is about 6 atoms, or about 5 to 7 atoms.

In yet other embodiments, the intramolecular bridge is formed betweentwo amino acids that are 6 amino acids apart, e.g., amino acids atpositions k and k+7, wherein k is any integer between 12 and 22 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22). In some specificembodiments, k is 12, 13, or 17. In an exemplary embodiment, k is 17.

Alpha Helix: Amino Acids Involved in Intramolecular Bridges

Examples of amino acid pairings that are capable of bonding (covalentlyor non-covalently) to form a six-atom linking bridge include Orn andAsp, Glu and an amino acid of Formula I, wherein n is 2, andhomoglutamic acid and an amino acid of Formula I, wherein n is 1,wherein Formula I is:

Examples of amino acid pairings that are capable of bonding to form aseven-atom linking bridge include Orn-Glu (lactam ring); Lys-Asp(lactam); or Homoser-Homoglu (lactone). Examples of amino acid pairingsthat may form an eight-atom linker include Lys-Glu (lactam); Homolys-Asp(lactam); Orn-Homoglu (lactam); 4-aminoPhe-Asp (lactam); or Tyr-Asp(lactone). Examples of amino acid pairings that may form a nine-atomlinker include Homolys-Glu (lactam); Lys-Homoglu (lactam);4-aminoPhe-Glu (lactam); or Tyr-Glu (lactone). Any of the side chains onthese amino acids may additionally be substituted with additionalchemical groups, so long as the three-dimensional structure of thealpha-helix is not disrupted. One of ordinary skill in the art canenvision alternative pairings or alternative amino acid analogs,including chemically modified derivatives, that would create astabilizing structure of similar size and desired effect. For example, ahomocysteine-homocysteine disulfide bridge is 6 atoms in length and maybe further modified to provide the desired effect.

Even without covalent linkage, the amino acid pairings described above(or similar pairings that one of ordinary skill in the art can envision)may also provide added stability to the alpha-helix through non-covalentbonds, for example, through formation of salt bridges orhydrogen-bonding interactions. Accordingly, salt bridges may be formedbetween: Orn and Glu; Lys and Asp; Homo-serine and Homo-glutamate; Lysand Glu; Asp and Arg; Homo-Lys and Asp; Orn and Homo-Glutamate;4-aminoPhe and Asp; Tyr and Asp; Homo-Lys and Glu; Lys and Homo-Glu;4-aminoPhe and Glu; or Tyr and Glu. In some embodiments, the analogcomprises a salt bridge between any of the following pairs of aminoacids: Orn and Glu; Lys and Asp; Lys and Glu; Asp and Arg; Homo-Lys andAsp; Orn and Homo-Glutamate; Homo-Lys and Glu; and Lys and Homo-Glu.Salt bridges may be formed between other pairs of oppositely chargedside chains. See, e.g., Kallenbach et al., Role of the Peptide Bond inProtein Structure and Folding, in The Amide Linkage: StructuralSignificance in Chemistry, Biochemistry, and Materials Science, JohnWiley & Sons, Inc. (2000).

In some embodiments, the non-covalent intramolecular bridge is ahydrophobic bridge. In accordance with one embodiment, the alpha helixof the analog is stabilized through the incorporation of hydrophobicamino acids at positions j and j+3 or i and i+4. For instance, i can beTyr and i+4 can be either Val or Leu; i can be Phe and i+4 can be Met;or i can be Phe and i+4 can be Ile. It should be understood that, forpurposes herein, the above amino acid pairings can be reversed, suchthat the indicated amino acid at position i could alternatively belocated at i+4, while the i+4 amino acid can be located at the iposition. It should also be understood that suitable amino acid pairingscan be formed for j and j+3.

Alpha Helix: Covalent Intramolecular Bridge

In some embodiments, the covalent intramolecular bridge is a lactam ringor lactam bridge. The size of the lactam ring can vary depending on thelength of the amino acid side chains, and in one embodiment the lactamis formed by linking the side chains of an ornithine to a aspartic acidside chain. Lactam bridges and methods of making the same are known inthe art. See, for example, Houston, Jr., et al., J Peptide Sci 1:274-282 (2004), and Example 1 herein. In some embodiments, the analogcomprises a modified sequence of SEQ ID NO: 1 and a lactam bridgebetween i and i+4, wherein i is as defined herein above. In someembodiments, the glucagon analog comprises two lactam bridges: onebetween the amino acids at positions 16 and 20 and another between theamino acids at positions 17 and 21. In some embodiments, the glucagonanalog comprises one lactam bridge and one salt bridge. Furtherexemplary embodiments, are described herein in the section entitled“EXAMPLES.” Further exemplary embodiments include the followingpairings, optionally with a lactam bridge: Glu at position 12 with Lysat position 16; native Lys at position 12 with Glu at position 16; Gluat position 16 with Lys at position 20; Lys at position 16 with Glu atposition 20; Glu at position 20 with Lys at position 24; Lys at position20 with Glu at position 24; Glu at position 24 with Lys at position 28;Lys at position 24 with Glu at position 28.

In some embodiments, the covalent intramolecular bridge is a lactone.Suitable methods of making a lactone bridge are known in the art. See,for example, Sheehan et al., J Am Chem Soc 95: 875-879 (1973).

In some aspects, olefin metathesis is used to cross-link one or twoturns of the alpha helix of the analog using an all-hydrocarboncross-linking system. The glucagon analog in this instance comprisesα-methylated amino acids bearing olefinic side chains of varying lengthand configured with either R or S stereochemistry at the j and j+3 or iand i+4 positions. In some embodiments, the olefinic side comprises(CH₂)n, wherein n is any integer between 1 to 6. In some embodiments, nis 3 for a cross-link length of 8 atoms. In some embodiments, n is 2 fora cross-link length of 6 atoms. An exemplary glucagon analog comprisingan olefinic cross-link is described herein as SEQ ID NO: 17. Suitablemethods of forming such intramolecular bridges are described in the art.See, for example, Schafmeister et al., J. Am. Chem. Soc. 122: 5891-5892(2000) and Walensky et al., Science 305: 1466-1470 (2004). Inalternative embodiments, the analog comprises O-allyl Ser residueslocated on adjacent helical turns, which are bridged together viaruthenium-catalyzed ring closing metathesis. Such procedures ofcross-linking are described in, for example, Blackwell et al., Angew,Chem., Int. Ed. 37: 3281-3284 (1998).

In specific aspects, use of the unnatural thio-dialanine amino acid,lanthionine, which has been widely adopted as a peptidomimetic ofcystine, is used to cross-link one turn of the alpha helix. Suitablemethods of lanthionine-based cyclization are known in the art. See, forinstance, Matteucci et al., Tetrahedron Letters 45: 1399-1401 (2004);Mayer et al., J. Peptide Res. 51: 432-436 (1998); Polinsky et al., J.Med. Chem. 35: 4185-4194 (1992); Osapay et al., J. Med. Chem. 40:2241-2251 (1997); Fukase et al., Bull. Chem. Soc. Jpn. 65: 2227-2240(1992); Harpp et al., J. Org. Chem. 36: 73-80 (1971); Goodman and Shao,Pure Appl. Chem. 68: 1303-1308 (1996); and Osapay and Goodman, J. Chem.Soc. Chem. Commun. 1599-1600 (1993).

In some embodiments, α,ω-diaminoalkane tethers, e.g., 1,4-diaminopropaneand 1,5-diaminopentane) between two Glu residues at positions i and i+7are used to stabilize the alpha helix of the analog. Such tethers leadto the formation of a bridge 9-atoms or more in length, depending on thelength of the diaminoalkane tether. Suitable methods of producingpeptides cross-linked with such tethers are described in the art. See,for example, Phelan et al., J. Am. Chem. Soc. 119: 455-460 (1997).

In yet other embodiments, a disulfide bridge is used to cross-link oneor two turns of the alpha helix of the analog. Alternatively, a modifieddisulfide bridge in which one or both sulfur atoms are replaced by amethylene group resulting in an isosteric macrocyclization is used tostabilize the alpha helix of the analog. Suitable methods of modifyingpeptides with disulfide bridges or sulfur-based cyclization aredescribed in, for example, Jackson et al., J. Am. Chem. Soc. 113:9391-9392 (1991) and Rudinger and Jost, Experientia 20: 570-571 (1964).

In yet other embodiments, the alpha helix of the analog is stabilizedvia the binding of metal atom by two His residues or a His and Cys pairpositioned at j and j+3, or i and i+4. The metal atom can be, forexample, Ru(III), Cu(II), Zn(II), or Cd(II). Such methods of metalbinding-based alpha helix stabilization are known in the art. See, forexample, Andrews and Tabor, Tetrahedron 55: 11711-11743 (1999); Ghadiriet al., J. Am. Chem. Soc. 112: 1630-1632 (1990); and Ghadiri et al., J.Am. Chem. Soc. 119: 9063-9064 (1997).

The alpha helix of the analog can alternatively be stabilized throughother means of peptide cyclizing, which means are reviewed in Davies, J.Peptide. Sci. 9: 471-501 (2003). The alpha helix can be stabilized viathe formation of an amide bridge, thioether bridge, thioester bridge,urea bridge, carbamate bridge, sulfonamide bridge, and the like. Forexample, a thioester bridge can be formed between the C-terminus and theside chain of a Cys residue. Alternatively, a thioester can be formedvia side chains of amino acids having a thiol (Cys) and a carboxylicacid (e.g., Asp, Glu). In another method, a cross-linking agent, such asa dicarboxylic acid, e.g., suberic acid (octanedioic acid), etc. canintroduce a link between two functional groups of an amino acid sidechain, such as a free amino, hydroxyl, thiol group, and combinationsthereof.

DPP-IV Resistant Peptides

In some embodiments, the glucagon analog comprises at position 1 or 2,or at both positions 1 and 2, an amino acid which achieves resistance ofthe glucagon analog to dipeptidyl peptidase IV (DPP IV) cleavage. Insome embodiments, the glucagon analog comprises at position 1 an aminoacid selected from the group consisting of: D-histidine,desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine,N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, oralpha, alpha-dimethyl imidiazole acetic acid (DMIA). In someembodiments, the glucagon analog comprises at position 2 an amino acidselected from the group consisting of: D-serine, D-alanine, valine,glycine, N-methyl serine, N-methyl alanine, or alpha, aminoisobutyricacid. In some embodiments, the glucagon analog comprises at position 2an amino acid which achieves resistance of the glucagon analog to DPP IVand the amino acid which achieves resistance of the glucagon analog toDPP IV is not D-serine.

In some aspects, the glucagon analog comprising an amino acid whichachieves resistance of the glucagon analog to DPP IV further comprisesan amino acid modification which stabilizes the alpha helix found in theC-terminal portion of glucagon, e.g., through a covalent bond betweenamino acids at positions “i” and “i+4”, e.g., 12 and 16, 16 and 20, or20 and 24. In some embodiments, this covalent bond is a lactam bridgebetween a glutamic acid at position 16 and a lysine at position 20. Insome embodiments, this covalent bond is an intramolecular bridge otherthan a lactam bridge. For example, suitable covalent bonding methodsinclude any one or more of olefin metathesis, lanthionine-basedcyclization, disulfide bridge or modified sulfur-containing bridgeformation, the use of a, w-diaminoalkane tethers, the formation ofmetal-atom bridges, and other means of peptide cyclization.

Modification of Position 1

In some specific embodiments, the glucagon analog comprises (a) an aminoacid substitution of His at position 1 with a large, aromatic amino acidand (b) an intramolecular bridge that stabilizes that alpha-helix in theC-terminal portion of the molecule (e.g., around positions 12-29). Inspecific embodiments, the amino acid at position 1 is replaced with Tyr,Phe, Trp, amino-Phe, nitro-Phe, chloro-Phe, sulfo-Phe, 4-pyridyl-Ala,methyl-Tyr, or 3-amino Tyr. The intramolecular bridge, in someembodiments, is any of those described herein. In some aspects, theintramolecular bridge is between the side chains of two amino acids thatare separated by three intervening amino acids, i.e., between the sidechains of amino acids i and i+4. In some embodiments, the intramolecularbridge is a lactam bridge. In some embodiments, the glucagon analogcomprises a large, aromatic amino acid at position 1 and a lactam bridgebetween the amino acids at positions 16 and 20 of the analog. Such aglucagon analog in some aspects further comprises one or more (e.g.,two, three, four, five or more) of the other modifications describedherein. For example, the glucagon analog can comprise an amide in placeof the C-terminal carboxylate. Also, in some embodiments, such glucagonanalogs further comprise one or more of a large aliphatic amino acid atposition 17, an imidazole containing amino acid at position 18, and apositive-charged amino acid at position 19. In some embodiments, theglucagon analogs comprising a modification at position 1 and anintramolecular bridge further comprises the amino acid sequenceIle-His-Gln at positions 17-19. Such modifications can be made withoutdestroying activity of the glucagon analog at the GLP-1 receptor and theglucagon receptor. In some embodiments, the glucagon analog additionallycomprises an acylated or alkylated amino acid residue.

Modification of Position 3

In some embodiments, the third amino acid of SEQ ID NO: 1 (Gln3) issubstituted with an acidic, basic, or hydrophobic amino acid residue andsuch modification causes the glucagon receptor activity to be reduced.In some embodiments, the acidic, basic, or hydrophobic amino acid isglutamic acid, ornithine, norleucine. In some aspects, modification withone of these residues has led the glucagon analog to exhibit asubstantially reduced or destroyed glucagon receptor activity. Theglucagon analogs that are substituted with, for example, glutamic acid,ornithine, or norleucine in some aspects have about 10% or less of theactivity of native glucagon at the glucagon receptor, e.g., about 1-10%,or about 0.1-10%, or greater than about 0.1% but less than about 10%,while exhibiting at least 20% of the activity of GLP-1 at the GLP-1receptor. In some embodiments, the glucagon analogs exhibit about 0.5%,about 1% or about 7% of the activity of native glucagon, whileexhibiting at least 20% of the activity of GLP-1 at the GLP-1 receptor.

In some embodiments, the glutamine at position 3 of SEQ ID NO: 1 of theglucagon analog is substituted with a glutamine analog without asubstantial loss of activity at the glucagon receptor, and in somecases, with an enhancement of glucagon receptor activity. In someembodiments, the glutamine analog is a naturally occurring or anon-naturally occurring or non-coded amino acid comprising a side chainof Structure I, II or III:

wherein R¹ is C₀₋₃ alkyl or C₀₋₃ heteroalkyl; R² is NHR⁴ or C₁₋₃ alkyl;R³ is C₁₋₃ alkyl; R⁴ is H or C₁₋₃ alkyl; X is NH, O, or S; and Y isNHR⁴, SR³, or OR³. In some embodiments, X is NH or Y is NHR⁴. In someembodiments, R¹ is C₀₋₂ alkyl or C₁ heteroalkyl. In some embodiments, R²is NHR⁴ or C₁ alkyl. In some embodiments, R⁴ is H or C¹ alkyl. Inexemplary embodiments, an amino acid comprising a side chain ofStructure I is provided where, R¹ is CH—S, X is NH, and R² is CH₃(acetamidomethyl-cysteine, C(Acm)); R¹ is CH₂, X is NH, and R² is CH₃(acetyldiaminobutanoic acid, Dab(Ac)); R¹ is C₀ alkyl, X is NH, R² isNHR⁴, and R⁴ is H (carbamoyldiaminopropanoic acid, Dap(urea)); or R¹ isCH₂—CH₂, X is NH, and R² is CH₃ (acetylornithine, Orn(Ac)). In exemplaryembodiments, an amino acid comprising a side chain of Structure II isprovide where, R¹ is CH₂, Y is NHR⁴, and R⁴ is CH₃ (methylglutamine,Q(Me)); In exemplary embodiments, an amino acid comprising a side chainof Structure III is provided where, R¹ is CH₂ and R⁴ is H(methionine-sulfoxide, M(O)); In specific embodiments, the amino acid atposition 3 is substituted with Dab(Ac) For example, glucagon agonistscan comprise a modified amino acid sequence of SEQ ID NO: 595, SEQ IDNO: 601 SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, and SEQ ID NO:606 of the sequence listing of International Patent Application No.PCT/US2009/047438, filed on Jun. 16, 2009, which is incorporated byreference in its entirety, wherein these amino acid sequences aremodified as further described herein, e.g., modified to comprise atleast three alpha helix promoting amino acids, modified to comprise (i)an acylated or alkylated amino acid at position 10, (ii) an alpha helixpromoting amino acid at position 16, (iii) an aliphatic amino acid atposition 17 and/or 18, and (iv) at least one charged amino acid locatedC-terminal to position 27, and, optionally, further modifications;modified to comprise at least three amino acids of the amino acids 18-24of Exendin-4 (SEQ ID NO: 8) at the corresponding positions of theglucaogon analog.

Modification of Position 7

In some embodiments, the glucagon analog comprises a modified SEQ ID NO:1 with an amino acid modification at position 7. In some aspects, theamino acid at position 7 of SEQ ID NO: 1 (Thr) is substituted with alarge, aliphatic amino acid, e.g., Ile, Leu, Ala, and the like. Suchmodifications are believed to drastically reduce activity at the GLP-1receptor of the glucagon analog.

Modification of Position 15

In some embodiments, the glucagon analogs comprise a modified SEQ ID NO:1 with an amino acid modification at position 15 which improvesstability. In some aspects, the amino acid at position 15 of SEQ ID NO:1 is deleted or substituted with glutamic acid, homoglutamic acid,cysteic acid or homocysteic acid. Such modifications reduce degradationor cleavage of the analog over time, especially in acidic or alkalinebuffers, e.g., buffers at a pH within the range of 5.5 to 8. In someembodiments, the glucagon analogs comprising this modification retainsat least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the original analogafter 24 hours at 25° C.

Modification of Position 16

In accordance with one embodiment, analogs of glucagon are provided thathave enhanced potency and optionally improved solubility and stability.In one embodiment, enhanced glucagon and GLP-1 potency is provided by anamino acid modification at position 16 of native glucagon (SEQ ID NO:1). By way of nonlimiting example, such enhanced potency can be providedby substituting the naturally occurring serine at position 16 withglutamic acid or with another negative-charged amino acid having a sidechain with a length of 4 atoms, or alternatively with any one ofglutamine, homoglutamic acid, or homocysteic acid, or a charged aminoacid having a side chain containing at least one heteroatom, (e.g., N,O, S, P) and with a side chain length of about 4 (or 3-5) atoms. In someembodiments, the glucagon analog comprises a modified SEQ ID NO: 1comprising a substitution of the Ser at position 16 with an amino acidselected from the group consisting of glutamic acid, glutamine,homoglutamic acid, homocysteic acid, threonine or glycine. In someaspects, the serine residue at position 16 is substituted with an aminoacid selected from the group consisting of glutamic acid, glutamine,homoglutamic acid and homocysteic acid. In some specific aspects, theserine residue at position 16 is substituted with glutamic acid or aconservative substitution thereof (e.g. an Exendin-4 amino acid).

In alternative embodiments, the glucagon analog comprises a modifiedsequence of SEQ ID NO: 1 modified by a substitution of Ser at position16 with Thr or AIB or another alpha helix promoting amino acid asdescribed above. In some embodiments, the alpha helix promoting aminoacid forms a non-covalent intramolecular bridge with an amino acid atj+3 or i+4.

Modification at Positions 17-18

In some embodiments, the glucagon analog comprises a modified SEQ ID NO:1 in which the dibasic Arg-Arg site at positions 17 and 18 iseliminated. Without being bound to any particular theory, it is believedthat elimination of the dibasic site in some embodiments improves the invivo efficacy of the glucagon analog. In some aspects, the glucagonanalog is modified in this regard by substituting one or both of theamino acids at positions 17 and 18 of SEQ ID NO: 1 with an amino acidwhich is not basic, e.g., with an aliphatic amino acid. In someembodiments, one of the amino acids at position 17 or 18 is deleted oran amino acid is inserted in between positions 17 and 18. In someembodiments, the Arg at position 17 is substituted with another aminoacid as described herein, e.g., Gln, an amino acid comprising ahydrophilic moiety, an alpha helix promoting amino acid. In someembodiments, the alpha helix promoting amino acid forms a non-covalentintramolecular bridge with an amino acid at j+3 or i+4. In someembodiments, the Arg at position 18 is substituted with another aminoacid as described herein. In exemplary aspects, the amino acid atposition 18 is an alpha, alpha, disubstituted amino acid, e.g., AIB. Insome aspects, the amino acid at position 18 is a small aliphatic aminoacid, e.g., Ala. In some specific aspects, the amino acid at position 18is a small aliphatic amino acid, e.g., Ala, and the Arg at position 17remains unmodified.

Modification of Position 20

Enhanced activity at the GLP-1 receptor is also provided by an aminoacid modification at position 20. In some embodiments, the glutamine atposition 20 is replaced with an alpha helix promoting amino acid, e.g.AIB, as described above. In some embodiments, the alpha helix promotingamino acid forms a non-covalent intramolecular bridge with an amino acidat j−3 or i−4. In some specific embodiments the amino acid is ahydrophilic amino acid having a side chain that is either charged or hasan ability to hydrogen-bond, and is at least about 5 (or about 4-6)atoms in length, for example, lysine, citrulline, arginine, orornithine, and optionally forms a salt bridge with another alpha helixpromoting amino acid at position 16, e.g. a negative charged amino acid.Such modifications in some particular aspects reduce degradation thatoccurs through deamidation of Gln and in some embodiments, increase theactivity of the glucagon analog at the GLP-1 receptor. In some aspects,the amino acid at position 20 is Glu or Lys or AIB.

Modification at Positions 21, 23, 24, and 28

In some embodiments, position 21 and/or position 24 is modified bysubstitution with an alpha helix promoting amino acid. In someembodiments, the alpha helix promoting amino acid forms a non-covalentintramolecular bridge with an amino acid at j−3 or i−4. In some aspects,the alpha helix promoting amino acid is AIB.

In exemplary embodiments, the amino acid at position 23 is a Ile.

In exemplary aspects, the amino acid at position 28 is an alpha, alpha,disubstituted amino acid, e.g., AIB.

Charged C-Terminus

In some embodiments, the glucagon analog is modified by amino acidsubstitutions and/or additions that introduce a charged amino acid intothe C-terminal portion of the analog. In some embodiments, suchmodifications enhance stability and solubility. As used herein the term“charged amino acid” or “charged residue” refers to an amino acid thatcomprises a side chain that is negative-charged (i.e., de-protonated) orpositive-charged (i.e., protonated) in aqueous solution at physiologicalpH. In some aspects, these amino acid substitutions and/or additionsthat introduce a charged amino acid modifications are at a positionC-terminal to position 27 of SEQ ID NO: 1. In some embodiments, one, twoor three (and in some instances, more than three) charged amino acidsare introduced within the C-terminal portion (e.g., position(s)C-terminal to position 27). In accordance with some embodiments, thenative amino acid(s) at positions 28 and/or 29 are substituted with acharged amino acids, and/or in a further embodiment one to three chargedamino acids are also added to the C-terminus of the analog. In exemplaryembodiments, one, two or all of the charged amino acids arenegative-charged. The negative-charged amino acid in some embodiments isaspartic acid, glutamic acid, cysteic acid, homocysteic acid, orhomoglutamic acid. In some aspects, these modifications increasesolubility, e.g., provide at least 2-fold, 5-fold, 10-fold, 15-fold,25-fold, 30-fold or greater solubility relative to native glucagon at agiven pH between about 5.5 and 8, e.g., pH 7, when measured after 24hours at 25° C.

C-Terminal Truncation

In accordance with some embodiments, the glucagon analogs disclosedherein are modified by truncation of the C-terminus by one or two aminoacid residues. Such modified glucagon peptides, as shown herein, retainsimilar activity and potency at the glucagon receptor and GLP-1receptor. In this regard, the glucagon peptides can comprise amino acids1-27 or 1-28 of the native glucagon analog (SEQ ID NO: 1), optionallywith any of the additional modifications described herein.

Charge-Neutral C-Terminus

In some embodiments, the glucagon analog comprises a modified SEQ ID NO:1 in which the carboxylic acid of the C-terminal amino acid is replacedwith a charge-neutral group, such as an amide or ester. Without beingbound to any particular theory, such modifications in certain aspectsincreases activity of the glucagon analog at the GLP-1 receptor.Accordingly, in some embodiments, the glucagon analog is an amidatedpeptide, such that the C-terminal residue comprises an amide in place ofthe alpha carboxylate of an amino acid. As used herein a generalreference to a peptide or analog is intended to encompass peptides thathave a modified amino terminus, carboxy terminus, or both amino andcarboxy termini. For example, an amino acid chain composing an amidegroup in place of the terminal carboxylic acid is intended to beencompassed by an amino acid sequence designating the standard aminoacids.

Other Modifications

In some embodiments, the glucagon analogs additionally or alternativelycomprise the following amino acid modifications:

-   -   (i) Substitution of Ser at position 2 with Ala;    -   (ii) Substitution of Tyr at position 10 with Val or Phe, or Trp;    -   (iii) Substitution of Lys at position 12 with Arg;    -   (iv) Substitution of Arg at position 17 with Gln or a small        aliphatic amino acid, e.g., Ala, or a large aliphatic amino        acid, e.g., Ile;    -   (v) Substitution of Arg at position 18 with a small aliphatic        amino acid, e.g., Ala; or an imidazole-containing amino acid,        e.g., His;    -   (vi) Substitution of Ala at position 19 with a positive-charged        amino acid, e.g., Gln;    -   (vii) Substitution of Val at position 23 with Ile, and    -   (viii) Substitution of Thr at position 29 with Gly or Gln.

In some embodiments, the stability of the glucagon analog is increasedby modification of the methionine at position 27, for example, bysubstitution with leucine or norleucine. Such modifications can reduceoxidative degradation. Stability can also be increased by modificationof the Gln at position 20 or 24 or 28, e.g., by substitution with Ala,Ser, Thr, or AIB. Such modifications can reduce degradation that occursthrough deamidation of Gln. Stability can be increased by modificationof Asp at position 21, e.g., by substitution with another acidicresidue, e.g., Glu. Such modifications can reduce degradation thatoccurs through dehydration of Asp to form a cyclic succinimideintermediate followed by isomerization to iso-aspartate.

In some embodiments, the glucagon analogs described herein areglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, cyclized via, e.g., a disulfide bridge, or converted into asalt (e.g., an acid addition salt, a basic addition salt), and/oroptionally dimerized, multimerized, or polymerized, or conjugated.

Any of the modifications described herein, including, for example, themodifications which increase or decrease glucagon receptor activity andwhich increase GLP-1 receptor activity, can be applied individually orin combination. Combinations of the modifications that increase GLP-1receptor activity may provide higher GLP-1 activity than any of suchmodifications taken alone.

EXEMPLARY EMBODIMENTS

The present disclosures provide peptides comprising a structure similarto that of native human glucagon and exhibiting enhanced agonistactivity at the GLP-1 receptor, compared to native human glucagon.Glucagon normally has about 1% of the activity of native-GLP-1 at theGLP-1 receptor, while GLP-1 normally has less than about 0.01% of theactivity of native glucagon at the glucagon receptor. Accordingly, thepeptides of the present disclosures exhibit greater than 1% of theactivity of native-GLP-1 at the GLP-1 receptor. In exemplaryembodiments, the peptides of the present disclosures exhibit greaterthan or about 5%, greater than or about 10%, greater than or about 15%,greater than or about 20%, greater than or about 25%, greater than orabout 30%, greater than or about 35%, greater than or about 40%, greaterthan or about 45%, greater than or about 50%, greater than or about 55%,greater than or about 60%, greater than or about 65%, greater than orabout 70%, greater than or about 75%, greater than or about 80%, greaterthan or about 85%, greater than or about 90%, or greater than or about95% of the activity of native-GLP-1 at the GLP-1 receptor. In exemplaryaspects, the peptides of the present disclosures exhibit activity at theGLP-1 receptor which is greater than that of native GLP-1. Accordingly,in exemplary aspects, the peptides of the present disclosures exhibitgreater than or about 100% of the activity of native-GLP-1 at the GLP-1receptor. In exemplary aspects, the peptides of the present disclosuresexhibit greater than or about 150%, greater than or about 200%, greaterthan or about 250%, greater than or about 300%, greater than or about350%, greater than or about 400%, greater than or about 450%, greaterthan or about 500%, greater than or about 550%, greater than or about600%, greater than or about 650%, greater than or about 700%, greaterthan or about 750%, greater than or about 800%, greater than or about850%, greater than or about 900%, greater than or about 950%, or greaterthan or about 1000% of the activity of native-GLP-1 at the GLP-1receptor.

In exemplary embodiments, the peptide comprises the amino acid sequenceof SEQ ID NO: 12.

In exemplary embodiments, the peptide comprises the amino acid sequenceof SEQ ID NO: 13.

In exemplary embodiments, the peptide comprises the amino acid sequenceof SEQ ID NO: 14.

In exemplary embodiments, the peptide comprises the amino acid sequenceof SEQ ID NO: 15.

In exemplary embodiments, the peptide comprises the amino acid sequenceof SEQ ID NO: 16 and exhibits at least 100-fold selectivity for thehuman GLP-1 receptor versus the GIP receptor.

In exemplary embodiments, the peptide comprises the amino acid sequenceof SEQ ID NO: 17.

The present disclosures further provides variant peptides comprising anamino acid sequence which is highly similar to the amino acid sequenceof one of the presently disclosed peptides. In exemplary embodiments,the variant peptide of the present disclosures comprises an amino acidsequence that is at least 80%, 85%, 90% or 95% identical to amino acids1-29 of the amino acid sequence of the peptide of any of SEQ ID NOs:12-17, wherein the variant peptide retains the activity of the parentpeptide at the GLP-1 receptor, glucagon receptor, and GIP receptor(e.g., exhibits at least 100-fold selectivity for the human GLP-1receptor versus the GIP receptor), and optionally a GLP-1 potency of atleast 1%; or wherein the EC50 of the peptide at the GIP receptor is lessthan 100-fold different from its EC50 at the GLP-1 receptor). Inexemplary embodiments, the variant peptide of the present disclosurescomprises exhibits at least 100-fold greater selectivity for the humanGLP-1 receptor versus the GIP receptor.

In exemplary embodiments, the variant peptide of the present disclosurescomprises an amino acid sequence based on an amino acid sequence of apeptide of the present disclosures but differs at one or more amino acidpositions, including, but not limited to position 1, position 2,position 3, position 7, position 10, position 12, position 15, position16, position 17, position 18, position 20, position 21, position 23,position 24, position 27, position 28, position 29. In exemplaryaspects, the variant peptide may comprise a conservative substitutionrelative to the parent peptide, may comprise any of the amino acidmodifications described herein, or may comprise an amino acidmodification that returns to the amino acid present at that position inthe native glucagon sequence (SEQ ID NO: 1). In exemplary aspects, thevariant peptide of the present disclosures comprises an amino acidsequence based on an amino acid sequence of a peptide of the presentdisclosures but differs in one or more of the following ways:

-   -   a) the variant peptide comprises an acylated amino acid or an        alkylated amino acid;    -   b) an acylated amino acid or an alkylated amino acid is replaced        with the corresponding amino acid of native glucagon (SEQ ID        NO: 1) at that position or a conservative substitution of the        native amino acid, and optionally a new acylated or alkylated        amino acid is introduced at a different position;    -   c) the variant peptide comprises an amino acid covalently        attached to a hydrophilic moiety;    -   d) an amino acid covalently attached to a hydrophilic moiety is        replaced with the corresponding amino acid of native glucagon        (SEQ ID NO: 1) at that position, and optionally a new amino acid        covalently attached to a hydrophilic moiety is introduced at a        different position;    -   e) the C-terminal amino acid of the variant peptide comprises a        C-terminal amide in place of a C-terminal alpha carboxylate;    -   f) an amino acid at any of positions 1 through 29 is replaced        with the corresponding amino acid of native glucagon (SEQ ID        NO: 1) at that position;    -   g) or any combinations thereof.

With regard to any of the foregoing variant peptides, in exemplaryembodiments, the variant peptide comprises a hydrophilic moietycovalently attached to an amino acid at position 16, 17, 21, 24, 29, aposition within a C-terminal extension, or at the C-terminus. Inexemplary aspects, the variant peptide comprises a Cys, Lys, Orn,homocysteine, and Ac-Phe covalently attached to a hydrophilic moiety,optionally, wherein the Cys, Lys, Orn, homocysteine, or Ac-Phe islocated at position 16, 17, 21, 24, 29, a position within a C-terminalextension, or at the C-terminus of the variant peptide. In exemplaryaspects, the hydrophilic moiety is a polyethylene glycol.

In exemplary aspects, the variant peptide comprises an acylated oralkylated amino acid, optionally, at position 10. In exemplary aspects,the variant peptide comprises an acylated or alkylated amino acid whichcomprises a C8 to C20 alkyl chain, a C12 to C18 alkyl chain, or a C14 orC16 alkyl chain. In exemplary aspects, the variant peptide comprises anacylated or alkylated amino acid which an acylated or alkylated aminoacid of Formula I, Formula II, or Formula III, optionally, wherein theamino acid of Formula I is Lys.

In exemplary aspects, the variant peptide of the present disclosurescomprises an acylated or alkylated amino acid, wherein the acyl group oralkyl group is covalently attached to the amino acid via a spacer,optionally, wherein the spacer is an amino acid or a dipeptide. Inexemplary embodiments, the spacer comprises one or two acidic residues.

In any of the foregoing exemplary embodiments, the peptide or variantpeptide of any of the present disclosures exhibits an (EC50 at theglucagon receptor)/(EC50 at the GLP-1 receptor) is about 20 or less(e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, 1, 0.5, 0.25, 0.10, 0.05, 0.025, 0.01, 0.001).

In any of the foregoing exemplary embodiments, the peptide or variantpeptide of any of the present disclosures exhibits an (EC50 at theglucagon receptor)/(EC50 at the GLP-1 receptor) is more than 20 (e.g.,21, 25, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1000, or more).

In any of the foregoing exemplary embodiments, the peptide or variantpeptide of any of the present disclosures exhibits an EC50 at the GLP-1receptor which is two- to ten-fold (e.g., 3-, 4-, 5-, 6-, 7-, 8-,9-fold) greater than the EC50 at the glucagon receptor.

Exclusions

In exemplary embodiments, any one of the following peptides is excludedfrom the glucagon analogs described herein, although any of thefollowing peptides comprising one or more further modifications theretoas described herein exhibiting the desired GLP-1 or co-agonist activity,pharmaceutical compositions, kits, and treatment methods using suchcompounds may be included in the invention: The peptide of SEQ ID NO: 1with an [Arg12] substitution and with a C-terminal amide; The peptide ofSEQ ID NO: 1 with [Arg12,Lys20] substitutions and with a C-terminalamide; The peptide of SEQ ID NO: 1 with [Arg12,Lys24] substitutions andwith a C-terminal amide; The peptide of SEQ ID NO: 1 with [Arg12,Lys29]substitutions and with a C-terminal amide; The peptide of SEQ ID NO: 1with a [Glu9] substitution; The peptide of SEQ ID NO: 1 missing His1,with [Glu9, Glu16, Lys29] substitutions and C-terminal amide; Thepeptide of SEQ ID NO: 1 with [Glu9, Glu16, Lys29] substitutions and witha C-terminal amide; The peptide of SEQ ID NO: 1 with [Lys13, Glu17]substitutions linked via lactam bridge and with a C-terminal amide; Thepeptide of SEQ ID NO: 1 with [Lys17, Glu21] substitutions linked vialactam bridge and with a C-terminal amide; The peptide of SEQ ID NO: 1missing His1, with [Glu20, Lys24] substitutions linked via lactambridge. In some embodiments, the glucagon analog is not any of thepeptides disclosed in any of International Patent Application No.PCT/US2009/034448, filed on Feb. 19, 2009, and published on Aug. 26,2010, as WO 2010/096052; International Patent Application No.PCT/US2009/068678, filed on Dec. 18, 2009, and published on Aug. 26,2010, as WO 2010/096142; International Patent Application No.PCT/US2009/047438, filed on Jun. 16, 2009, and published on Dec. 23,2009 as WO 2009/155258; International Patent Application No.PCT/US2008/053857, filed on Feb. 13, 2008, and published on Aug. 21,2008, as WO 2008/101017; International Patent Application No.PCT/US2010/059724, filed on Dec. 9, 2010; International PatentApplication No. PCT/US2009/047447, filed on Jun. 16, 2009, and publishedon Jan. 28, 2010, as WO2010/011439; International Patent Application No.PCT/US2010/38825, filed on Jun. 16, 2010, and published on Dec. 23,2010, as WO2010/148089; International Patent Application No.PCT/US2011/022608, filed on Jan. 26, 2011; and U.S. ProvisionalApplication No. 61/426,285, filed on Dec. 22, 2010; each of which areincorporated by reference in their entirety. In some embodiments, theglucagon analog does not include all or part of the sequence KRNRNNIAlinked to the C-terminus after position 29, e.g. KRNR.

Methods of Making Peptides

The glucagon analogs of the disclosure can be obtained by methods knownin the art. Suitable methods of de novo synthesizing peptides aredescribed in, for example, Chan et al., Fmoc Solid Phase PeptideSynthesis, Oxford University Press, Oxford, United Kingdom, 2005;Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc.,2000; Epitope Mapping, ed. Westwood et al., Oxford University Press,Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752.

Also, in the instances in which the analogs of the disclosure do notcomprise any non-coded or non-natural amino acids, the glucagon analogcan be recombinantly produced using a nucleic acid encoding the aminoacid sequence of the analog using standard recombinant methods. See, forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, N Y, 1994.

In some embodiments, the glucagon analogs of the disclosure areisolated. In some embodiments, the glucagon analogs of the disclosureare purified. It is recognized that “purity” is a relative term, and notto be necessarily construed as absolute purity or absolute enrichment orabsolute selection. In some aspects, the purity is at least or about50%, is at least or about 60%, at least or about 70%, at least or about80%, or at least or about 90% (e.g., at least or about 91%, at least orabout 92%, at least or about 93%, at least or about 94%, at least orabout 95%, at least or about 96%, at least or about 97%, at least orabout 98%, at least or about 99% or is approximately 100%.

In some embodiments, the peptides described herein are commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the peptides can be synthetic,recombinant, isolated, and/or purified.

Conjugates

The invention further provides conjugates comprising one or more of theglucagon analogs described herein conjugated to a heterologous moiety,wherein the conjugate exhibits enhanced activity at the GLP-1 receptor,as compared to native glucagon, and exhibits at least 100-fold greaterselectivity for the human GLP-1 receptor versus the GIP receptor. Asused herein, the term “heterologous moiety” is synonymous with the term“conjugate moiety” and refers to any molecule (chemical or biochemical,naturally-occurring or non-coded) which is different from the glucagonanalogs described herein. Exemplary conjugate moieties that can belinked to any of the analogs described herein include but are notlimited to a heterologous peptide or polypeptide (including for example,a plasma protein), a targeting agent, an immunoglobulin or portionthereof (e.g., variable region, CDR, or Fc region), a diagnostic labelsuch as a radioisotope, fluorophore or enzymatic label, a polymerincluding water soluble polymers, or other therapeutic or diagnosticagents. In some embodiments a conjugate is provided comprising an analogof the present invention and a plasma protein, wherein the plasmaprotein is selected from the group consisting of albumin, transferin,fibrinogen and globulins. In some embodiments the plasma protein moietyof the conjugate is albumin or transferin. The conjugate in someembodiments comprises one or more of the glucagon analogs describedherein and one or more of: a peptide (which is distinct from theglucagon and/or GLP-1 receptor active glucagon analogs describedherein), a polypeptide, a nucleic acid molecule, an antibody or fragmentthereof, a polymer, a quantum dot, a small molecule, a toxin, adiagnostic agent, a carbohydrate, an amino acid.

In some embodiments, the heterologous moiety is a peptide which isdistinct from the glucagon and/or GLP-1 receptor active analogsdescribed herein and the conjugate is a fusion peptide or a chimericpeptide. In some embodiments, the heterologous moiety is a peptideextension of 1-21 amino acids. In specific embodiments, the extension isattached to the C-terminus of the glucagon analog, e.g., to amino acidat position 29.

In some specific aspects, the extension is a single amino acid ordipeptide. In specific embodiments, the extension comprises an aminoacid selected from the group consisting of: a charged amino acid (e.g.,a negative-charged amino acid (e.g., Glu), a positive-charged aminoacid), an amino acid comprising a hydrophilic moiety. In some aspects,the extension is Gly, Glu, Cys, Gly-Gly, Gly-Glu.

In some embodiments, the extension comprises an amino acid sequence ofSEQ ID NO: 9 (GPSSGAPPPS), SEQ ID NO: 10 (GGPSSGAPPPS), SEQ ID NO: 8(KRNRNNIA), or SEQ ID NO: 11 (KRNR). In specific aspects, the amino acidsequence is attached through the C-terminal amino acid of the glucagonanalog, e.g., amino acid at position 29. In some embodiments, the aminoacid sequence of SEQ ID NOs: 13-16 is bound to amino acid 29 of theglucagon analog through a peptide bond. In some specific embodiments,the amino acid at position 29 of the glucagon analog is a Gly and theGly is fused to one of the amino acid sequences of SEQ ID NOs: 8-11.

In some embodiments, the heterologous moiety is a polymer. In someembodiments, the polymer is selected from the group consisting of:polyamides, polycarbonates, polyalkylenes and derivatives thereofincluding, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polymers of acrylic and methacrylic esters, includingpoly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene.

In some aspects, the polymer is a biodegradable polymer, including asynthetic biodegradable polymer (e.g., polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone)),and a natural biodegradable polymer (e.g., alginate and otherpolysaccharides including dextran and cellulose, collagen, chemicalderivatives thereof (substitutions, additions of chemical groups, forexample, alkyl, alkylene, hydroxylations, oxidations, and othermodifications routinely made by those skilled in the art), albumin andother hydrophilic proteins (e.g., zein and other prolamines andhydrophobic proteins)), as well as any copolymer or mixture thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

In some aspects, the polymer is a bioadhesive polymer, such as abioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A.Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer or ahydrophilic polymer. Hydrophilic polymers are further described hereinunder “Hydrophilic Moieties.” Suitable water-soluble polymers are knownin the art and include, for example, polyvinylpyrrolidone, hydroxypropylcellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel),nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropylbutylcellulose, hydroxypropyl pentylcellulose, methyl cellulose,ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkylcelluloses and hydroxyalkyl celluloses, various cellulose ethers,cellulose acetate, carboxymethyl cellulose, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acidcopolymers, poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate,methacrylic acid copolymers, polymethacrylic acid,polymethylmethacrylate, maleic anhydride/methyl vinyl ether copolymers,poly vinyl alcohol, sodium and calcium polyacrylic acid, polyacrylicacid, acidic carboxy polymers, carboxypolymethylene, carboxyvinylpolymers, polyoxyethylene polyoxypropylene copolymer,polymethylvinylether co-maleic anhydride, carboxymethylamide, potassiummethacrylate divinylbenzene co-polymer, polyoxyethyleneglycols,polyethylene oxide, and derivatives, salts, and combinations thereof.

In specific embodiments, the polymer is a polyalkylene glycol,including, for example, polyethylene glycol (PEG).

In some embodiments, the heterologous moiety is a carbohydrate. In someembodiments, the carbohydrate is a monosaccharide (e.g., glucose,galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose),an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (astarch, amylase, amylopectin, cellulose, chitin, callose, laminarin,xylan, mannan, fucoidan, galactomannan.

In some embodiments, the heterologous moiety is a lipid. The lipid, insome embodiments, is a fatty acid, eicosanoid, prostaglandin,leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g.,mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin,monoglyceride, diglyceride, triglyceride, a phospholipid.

In some embodiments, the heterologous moiety is attached vianon-covalent or covalent bonding to the analog of the presentdisclosure. In certain aspects, the heterologous moiety is attached tothe analog of the present disclosure via a linker. Linkage can beaccomplished by covalent chemical bonds, physical forces suchelectrostatic, hydrogen, ionic, van der Waals, or hydrophobic orhydrophilic interactions. A variety of non-covalent coupling systems maybe used, including biotin-avidin, ligand/receptor, enzyme/substrate,nucleic acid/nucleic acid binding protein, lipid/lipid binding protein,cellular adhesion molecule partners; or any binding partners orfragments thereof which have affinity for each other.

The glucagon analog in some embodiments is linked to conjugate moietiesvia direct covalent linkage by reacting targeted amino acid residues ofthe analog with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthese targeted amino acids. Reactive groups on the analog or conjugatemoiety include, e.g., an aldehyde, amino, ester, thiol, α-haloacetyl,maleimido or hydrazino group. Derivatizing agents include, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride or other agents known in the art.Alternatively, the conjugate moieties can be linked to the analogindirectly through intermediate carriers, such as polysaccharide orpolypeptide carriers. Examples of polysaccharide carriers includeaminodextran. Examples of suitable polypeptide carriers includepolylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof,and mixed polymers of these amino acids and others, e.g., serines, toconfer desirable solubility properties on the resultant loaded carrier.

Cysteinyl residues are most commonly reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid, chloroacetamide togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,alpha-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),deamidation of asparagine or glutamine, acetylation of the N-terminalamine, and/or amidation or esterification of the C-terminal carboxylicacid group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the analog. Sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of tyrosine, or tryptophan, or (f) theamide group of glutamine. These methods are described in WO87/05330published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

In some embodiments, the glucagon analog is conjugated to a heterologousmoiety via covalent linkage between a side chain of an amino acid of theglucagon analog and the heterologous moiety. In some embodiments, theglucagon analog is conjugated to a heterologous moiety via the sidechain of an amino acid at position 16, 17, 21, 24, or 29, a positionwithin a C-terminal extension, or the C-terminal amino acid, or acombination of these positions. In some aspects, the amino acidcovalently linked to a heterologous moiety (e.g., the amino acidcomprising a heterologous moiety) is a Cys, Lys, Orn, homo-Cys, orAc-Phe, and the side chain of the amino acid is covalently bonded to aheterologous moiety.

In some embodiments, the conjugate comprises a linker that joins theglucagon analog to the heterologous moiety. In some aspects, the linkercomprises a chain of atoms from 1 to about 60, or 1 to 30 atoms orlonger, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atomslong. In some embodiments, the chain atoms are all carbon atoms. In someembodiments, the chain atoms in the backbone of the linker are selectedfrom the group consisting of C, O, N, and S. Chain atoms and linkers maybe selected according to their expected solubility (hydrophilicity) soas to provide a more soluble conjugate. In some embodiments, the linkerprovides a functional group that is subject to cleavage by an enzyme orother catalyst or hydrolytic conditions found in the target tissue ororgan or cell. In some embodiments, the length of the linker is longenough to reduce the potential for steric hindrance. If the linker is acovalent bond or a peptidyl bond and the conjugate is a polypeptide, theentire conjugate can be a fusion protein. Such peptidyl linkers may beany length. Exemplary linkers are from about 1 to 50 amino acids inlength, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids inlength. Such fusion proteins may alternatively be produced byrecombinant genetic engineering methods known to one of ordinary skillin the art.

Conjugates: Fc Fusions

As noted above, in some embodiments, the analogs are conjugated, e.g.,fused to an immunoglobulin or portion thereof (e.g., variable region,CDR, or Fc region). Known types of immunoglobulins (Ig) include IgG,IgA, IgE, IgD or IgM. The Fc region is a C-terminal region of an Igheavy chain, which is responsible for binding to Fc receptors that carryout activities such as recycling (which results in prolonged half-life),antibody dependent cell-mediated cytotoxicity (ADCC), and complementdependent cytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. In a relatedembodiments, the Fc region may comprise one or more native or modifiedconstant regions from an immunoglobulin heavy chain, other than CH1, forexample, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4regions of IgE.

Suitable conjugate moieties include portions of immunoglobulin sequencethat include the FcRn binding site. FcRn, a salvage receptor, isresponsible for recycling immunoglobulins and returning them tocirculation in blood. The region of the Fc portion of IgG that binds tothe FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not include FcγR binding site(s).FcγR are responsible for ADCC and CDC. Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(lower hinge region), amino acids 265-269 (B/C loop), amino acids297-299 (C′/E loop), and amino acids 327-332 (F/G) loop (Sondermann etal., Nature 406: 267-273, 2000). The lower hinge region of IgE has alsobeen implicated in the FcRI binding (Henry, et al., Biochemistry 36,15568-15578, 1997). Residues involved in IgA receptor binding aredescribed in Lewis et al., (J Immunol. 175:6694-701, 2005). Amino acidresidues involved in IgE receptor binding are described in Sayers et al.(J Biol Chem. 279(34):35320-5, 2004).

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγRs (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγRs(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, eachincorporated by reference herein in its entirety.

Conjugates: Hydrophilic Moieties

The glucagon analogs described herein can be further modified to improveits solubility and stability in aqueous solutions at physiological pH,while retaining the high biological activity relative to nativeglucagon. Hydrophilic moieties such as PEG groups can be attached to theanalogs under any suitable conditions used to react a protein with anactivated polymer molecule. Any means known in the art can be used,including via acylation, reductive alkylation, Michael addition, thiolalkylation or other chemoselective conjugation/ligation methods througha reactive group on the PEG moiety (e.g., an aldehyde, amino, ester,thiol, α-haloacetyl, maleimido or hydrazino group) to a reactive groupon the target compound (e.g., an aldehyde, amino, ester, thiol,α-haloacetyl, maleimido or hydrazino group). Activating groups which canbe used to link the water soluble polymer to one or more proteinsinclude without limitation sulfone, maleimide, sulfhydryl, thiol,triflate, tresylate, azidirine, oxirane, 5-pyridyl, andalpha-halogenated acyl group (e.g., alpha-iodo acetic acid,alpha-bromoacetic acid, alpha-chloroacetic acid). If attached to theanalog by reductive alkylation, the polymer selected should have asingle reactive aldehyde so that the degree of polymerization iscontrolled. See, for example, Kinstler et al., Adv. Drug. Delivery Rev.54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459-476(2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16: 157-182 (1995).

In specific aspects, an amino acid residue of the analog having a thiolis modified with a hydrophilic moiety such as PEG. In some embodiments,the thiol is modified with maleimide-activated PEG in a Michael additionreaction to result in a PEGylated analog comprising the thioetherlinkage shown below:

In some embodiments, the thiol is modified with a haloacetyl-activatedPEG in a nucleophilic substitution reaction to result in a PEGylatedanalog comprising the thioether linkage shown below:

Suitable hydrophilic moieties include polyethylene glycol (PEG),polypropylene glycol, polyoxyethylated polyols (e.g., POG),polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol (POG), polyoxyalkylenes, polyethylene glycol propionaldehyde,copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethyleneglycol, mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol,carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, poly (.beta.-amino acids) (either homopolymers orrandom copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers (PPG) and other polyakylene oxides,polypropylene oxide/ethylene oxide copolymers, colonic acids or otherpolysaccharide polymers, Ficoll or dextran and mixtures thereof.Dextrans are polysaccharide polymers of glucose subunits, predominantlylinked by α1-6 linkages. Dextran is available in many molecular weightranges, e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD. Linear or branchedpolymers are contemplated. Resulting preparations of conjugates may beessentially monodisperse or polydisperse, and may have about 0.5, 0.7,1, 1.2, 1.5 or 2 polymer moieties per analog.

In some embodiments, the glucagon analog is conjugated to a hydrophilicmoiety via covalent linkage between a side chain of an amino acid of theglucagon analog and the hydrophilic moiety. In some embodiments, theglucagon analog is conjugated to a hydrophilic moiety via the side chainof an amino acid at position 16, 17, 21, 24, or 29, a position within aC-terminal extension, or the C-terminal amino acid, or a combination ofthese positions. In some aspects, the amino acid covalently linked to ahydrophilic moiety (e.g., the amino acid comprising a hydrophilicmoiety) is a Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the side chain ofthe amino acid is covalently bonded to a hydrophilic moiety (e.g., PEG).

Conjugates: rPEG

In some embodiments, the conjugate of the present disclosure comprisesthe analog having glucagon and/or GLP-1 agonist activity fused to anaccessory analog which is capable of forming an extended conformationsimilar to chemical PEG (e.g., a recombinant PEG (rPEG) molecule), suchas those described in International Patent Application Publication No.WO2009/023270 and U.S. Patent Application Publication No. US20080286808.The rPEG molecule in some aspects is a polypeptide comprising one ormore of glycine, serine, glutamic acid, aspartic acid, alanine, orproline. In some aspects, the rPEG is a homopolymer, e.g., poly-glycine,poly-serine, poly-glutamic acid, poly-aspartic acid, poly-alanine, orpoly-proline. In other embodiments, the rPEG comprises two types ofamino acids repeated, e.g., poly(Gly-Ser), poly(Gly-Glu), poly(Gly-Ala),poly(Gly-Asp), poly(Gly-Pro), poly(Ser-Glu), etc. In some aspects, therPEG comprises three different types of amino acids, e.g.,poly(Gly-Ser-Glu). In specific aspects, the rPEG increases the half-lifeof the Glucagon and/or GLP-1 agonist analog. In some aspects, the rPEGcomprises a net positive or net negative charge. The rPEG in someaspects lacks secondary structure. In some embodiments, the rPEG isgreater than or equal to 10 amino acids in length and in someembodiments is about 40 to about 50 amino acids in length. The accessorypeptide in some aspects is fused to the N- or C-terminus of the analogof the present disclosure through a peptide bond or a proteinasecleavage site, or is inserted into the loops of the analog of thepresent disclosure. The rPEG in some aspects comprises an affinity tagor is linked to a PEG that is greater than 5 kDa. In some embodiments,the rPEG confers the analog of the present disclosure with an increasedhydrodynamic radius, serum half-life, protease resistance, or solubilityand in some aspects confers the analog with decreased immunogenicity.

Conjugates: Multimers

The invention further provides multimers or dimers of the analogsdisclosed herein, including homo- or hetero-multimers or homo- orhetero-dimers. Two or more of the analogs can be linked together usingstandard linking agents and procedures known to those skilled in theart. For example, dimers can be formed between two peptides through theuse of bifunctional thiol crosslinkers and bi-functional aminecrosslinkers, particularly for the analogs that have been substitutedwith cysteine, lysine ornithine, homocysteine or acetyl phenylalanineresidues. The dimer can be a homodimer or alternatively can be aheterodimer. In certain embodiments, the linker connecting the two (ormore) analogs is PEG, e.g., a 5 kDa PEG, 20 kDa PEG. In someembodiments, the linker is a disulfide bond. For example, each monomerof the dimer may comprise a Cys residue (e.g., a terminal or internallypositioned Cys) and the sulfur atom of each Cys residue participates inthe formation of the disulfide bond. In some aspects, the monomers areconnected via terminal amino acids (e.g., N-terminal or C-terminal), viainternal amino acids, or via a terminal amino acid of at least onemonomer and an internal amino acid of at least one other monomer. Inspecific aspects, the monomers are not connected via an N-terminal aminoacid. In some aspects, the monomers of the multimer are attachedtogether in a “tail-to-tail” orientation in which the C-terminal aminoacids of each monomer are attached together.

Pharmaceutical Compositions, Uses and Kits

Salts

In some embodiments, the glucagon analog is in the form of a salt, e.g.,a pharmaceutically acceptable salt. As used herein the term“pharmaceutically acceptable salt” refers to salts of compounds thatretain the biological activity of the parent compound, and which are notbiologically or otherwise undesirable. Such salts can be prepared insitu during the final isolation and purification of the analog, orseparately prepared by reacting a free base function with a suitableacid. Many of the compounds disclosed herein are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Representative acid addition salts include,but are not limited to acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, sodium bisulfate, butyrate,camphorate, camphor sulfonate, digluconate, glycerophosphate,hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate),lactate, maleate, methane sulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitoate, pectinate, persulfate,3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate,tartrate, thiocyanate, phosphate, glutamate, bicarbonate,p-toluenesulfonate, and undecanoate. Salts derived from inorganic acidsinclude hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, trifluoroacetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid,maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluene-sulfonic acid, salicylic acid, and the like. Examples of acidswhich can be employed to form pharmaceutically acceptable acid additionsalts include, for example, an inorganic acid, e.g., hydrochloric acid,hydrobromic acid, sulphuric acid, and phosphoric acid, and an organicacid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.

Basic addition salts also can be prepared in situ during the finalisolation and purification of the source of salicylic acid, or byreacting a carboxylic acid-containing moiety with a suitable base suchas the hydroxide, carbonate, or bicarbonate of a pharmaceuticallyacceptable metal cation or with ammonia or an organic primary,secondary, or tertiary amine. Pharmaceutically acceptable salts include,but are not limited to, cations based on alkali metals or alkaline earthmetals such as lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like, and nontoxic quaternary ammonia and aminecations including ammonium, tetramethylammonium, tetraethylammonium,methylammonium, dimethylammonium, trimethylammonium, triethylammonium,diethylammonium, and ethylammonium, amongst others. Other representativeorganic amines useful for the formation of base addition salts include,for example, ethylenediamine, ethanolamine, diethanolamine, piperidine,piperazine, and the like. Salts derived from organic bases include, butare not limited to, salts of primary, secondary and tertiary amines.

Further, basic nitrogen-containing groups can be quaternized with theanalog of the present disclosure as lower alkyl halides such as methyl,ethyl, propyl, and butyl chlorides, bromides, and iodides; long chainhalides such as decyl, lauryl, myristyl, and stearyl chlorides,bromides, and iodides; arylalkyl halides like benzyl and phenethylbromides and others. Water or oil-soluble or dispersible products arethereby obtained.

Formulations

In accordance with some embodiments, a pharmaceutical composition isprovided wherein the composition comprises a glucagon analog of thepresent disclosure, or pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. The pharmaceutical composition cancomprise any pharmaceutically acceptable ingredient, including, forexample, acidifying agents, additives, adsorbents, aerosol propellants,air displacement agents, alkalizing agents, anticaking agents,anticoagulants, antimicrobial preservatives, antioxidants, antiseptics,bases, binders, buffering agents, chelating agents, coating agents,coloring agents, desiccants, detergents, diluents, disinfectants,disintegrants, dispersing agents, dissolution enhancing agents, dyes,emollients, emulsifying agents, emulsion stabilizers, fillers, filmforming agents, flavor enhancers, flavoring agents, flow enhancers,gelling agents, granulating agents, humectants, lubricants,mucoadhesives, ointment bases, ointments, oleaginous vehicles, organicbases, pastille bases, pigments, plasticizers, polishing agents,preservatives, sequestering agents, skin penetrants, solubilizingagents, solvents, stabilizing agents, suppository bases, surface activeagents, surfactants, suspending agents, sweetening agents, therapeuticagents, thickening agents, tonicity agents, toxicity agents,viscosity-increasing agents, water-absorbing agents, water-misciblecosolvents, water softeners, or wetting agents.

In some embodiments, the pharmaceutical composition comprises any one ora combination of the following components: acacia, acesulfame potassium,acetyltributyl citrate, acetyltriethyl citrate, agar, albumin, alcohol,dehydrated alcohol, denatured alcohol, dilute alcohol, aleuritic acid,alginic acid, aliphatic polyesters, alumina, aluminum hydroxide,aluminum stearate, amylopectin, α-amylose, ascorbic acid, ascorbylpalmitate, aspartame, bacteriostatic water for injection, bentonite,bentonite magma, benzalkonium chloride, benzethonium chloride, benzoicacid, benzyl alcohol, benzyl benzoate, bronopol, butylatedhydroxyanisole, butylated hydroxytoluene, butylparaben, butylparabensodium, calcium alginate, calcium ascorbate, calcium carbonate, calciumcyclamate, dibasic anhydrous calcium phosphate, dibasic dehydratecalcium phosphate, tribasic calcium phosphate, calcium propionate,calcium silicate, calcium sorbate, calcium stearate, calcium sulfate,calcium sulfate hemihydrate, canola oil, carbomer, carbon dioxide,carboxymethyl cellulose calcium, carboxymethyl cellulose sodium,β-carotene, carrageenan, castor oil, hydrogenated castor oil, cationicemulsifying wax, cellulose acetate, cellulose acetate phthalate, ethylcellulose, microcrystalline cellulose, powdered cellulose, silicifiedmicrocrystalline cellulose, sodium carboxymethyl cellulose, cetostearylalcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol,chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidinegluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC),chlorodifluoromethane, chlorofluorocarbons (CFC) chlorophenoxyethanol,chloroxylenol, corn syrup solids, anhydrous citric acid, citric acidmonohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil,cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium,crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin,dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate,dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane(HFC), dimethyl-β-cyclodextrin, cyclodextrin-type compounds such asCaptisol®, dimethyl ether, dimethyl phthalate, dipotassium edentate,disodium edentate, disodium hydrogen phosphate, docusate calcium,docusate potassium, docusate sodium, dodecyl gallate,dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid,eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate,ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium,ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructosemilled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin,glucose, liquid glucose, glyceride mixtures of saturated vegetable fattyacids, glycerin, glyceryl behenate, glyceryl monooleate, glycerylmonostearate, self-emulsifying glyceryl monostearate, glycerylpalmitostearate, glycine, glycols, glycofurol, guar gum,heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, highfructose syrup, human serum albumin, hydrocarbons (HC), dilutehydrochloric acid, hydrogenated vegetable oil, type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,imidurea, indigo carmine, ion exchangers, iron oxides, isopropylalcohol, isopropyl myristate, isopropyl palmitate, isotonic saline,kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols,anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesiumcarbonate, normal magnesium carbonate, magnesium carbonate anhydrous,magnesium carbonate hydroxide, magnesium hydroxide, magnesium laurylsulfate, magnesium oxide, magnesium silicate, magnesium stearate,magnesium trisilicate, magnesium trisilicate anhydrous, malic acid,malt, maltitol, maltitol solution, maltodextrin, maltol, maltose,mannitol, medium chain triglycerides, meglumine, menthol,methylcellulose, methyl methacrylate, methyl oleate, methylparaben,methylparaben potassium, methylparaben sodium, microcrystallinecellulose and carboxymethylcellulose sodium, mineral oil, light mineraloil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine,montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin,peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceuticalglaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer,polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates,polyethylene-polyoxypropylene-block polymers, polymethacrylates,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates,polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassiumbenzoate, potassium bicarbonate, potassium bisulfite, potassiumchloride, potassium citrate, potassium citrate anhydrous, potassiumhydrogen phosphate, potassium metabisulfite, monobasic potassiumphosphate, potassium propionate, potassium sorbate, povidone, propanol,propionic acid, propylene carbonate, propylene glycol, propylene glycolalginate, propyl gallate, propylparaben, propylparaben potassium,propylparaben sodium, protamine sulfate, rapeseed oil, Ringer'ssolution, saccharin, saccharin ammonium, saccharin calcium, saccharinsodium, safflower oil, saponite, serum proteins, sesame oil, colloidalsilica, colloidal silicon dioxide, sodium alginate, sodium ascorbate,sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride,anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride,sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium laurylsulfate, sodium metabisulfite, sodium phosphate, dibasic, sodiumphosphate, monobasic, sodium phosphate, tribasic, anhydrous sodiumpropionate, sodium propionate, sodium sorbate, sodium starch glycolate,sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters(sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil,spermaceti wax, starch, corn starch, potato starch, pregelatinizedstarch, sterilizable maize starch, stearic acid, purified stearic acid,stearyl alcohol, sucrose, sugars, compressible sugar, confectioner'ssugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF,synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane(HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol,tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol,delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributylcitrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin,trimethyltetradecylammonium bromide, tris buffer, trisodium edentate,vanillin, type I hydrogenated vegetable oil, water, soft water, hardwater, carbon dioxide-free water, pyrogen-free water, water forinjection, sterile water for inhalation, sterile water for injection,sterile water for irrigation, waxes, anionic emulsifying wax, carnaubawax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax,nonionic emulsifying wax, suppository wax, white wax, yellow wax, whitepetrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zincsalts, zinc stearate, or any excipient in the Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), which is incorporated byreference in its entirety, discloses various components used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional agent is incompatible with the pharmaceutical compositions,its use in pharmaceutical compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

In some embodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration, such as, for example,at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v,1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60%w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the foregoingcomponent(s) may be present in the pharmaceutical composition at anyconcentration, such as, for example, at most B, wherein B is 90% w/v,80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v,5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In otherembodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration range, such as, forexample from about A to about B. In some embodiments, A is 0.0001% and Bis 90%.

The pharmaceutical compositions may be formulated to achieve aphysiologically compatible pH. In some embodiments, the pH of thepharmaceutical composition may be at least 5, at least 5.5, at least 6,at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, atleast 9, at least 9.5, at least 10, or at least 10.5 up to and includingpH 11, depending on the formulation and route of administration. Incertain embodiments, the pharmaceutical compositions may comprisebuffering agents to achieve a physiological compatible pH. The bufferingagents may include any compounds capable of buffering at the desired pHsuch as, for example, phosphate buffers (e.g., PBS), triethanolamine,Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES,and others. In certain embodiments, the strength of the buffer is atleast 0.5 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 20mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, atleast 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least120 mM, at least 150 mM, or at least 200 mM. In some embodiments, thestrength of the buffer is no more than 300 mM (e.g., at most 200 mM, atmost 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM,at most 50 mM, at most 40 mM, at most 30 mM, at most 20 mM, at most 10mM, at most 5 mM, at most 1 mM).

Routes of Administration

The following discussion on routes of administration is merely providedto illustrate exemplary embodiments and should not be construed aslimiting the scope in any way.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the analog of the presentdisclosure dissolved in diluents, such as water, saline, or orangejuice; (b) capsules, sachets, tablets, lozenges, and troches, eachcontaining a predetermined amount of the active ingredient, as solids orgranules; (c) powders; (d) suspensions in an appropriate liquid; and (e)suitable emulsions. Liquid formulations may include diluents, such aswater and alcohols, for example, ethanol, benzyl alcohol, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant. Capsule forms can be of theordinary hard- or soft-shelled gelatin type containing, for example,surfactants, lubricants, and inert fillers, such as lactose, sucrose,calcium phosphate, and corn starch. Tablet forms can include one or moreof lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the analog of the presentdisclosure in a flavor, usually sucrose and acacia or tragacanth, aswell as pastilles comprising the analog of the present disclosure in aninert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to, suchexcipients as are known in the art.

The analogs of the disclosure, alone or in combination with othersuitable components, can be delivered via pulmonary administration andcan be made into aerosol formulations to be administered via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also may be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations also may be used to spray mucosa. In some embodiments, theanalog is formulated into a powder blend or into microparticles ornanoparticles. Suitable pulmonary formulations are known in the art.See, e.g., Qian et al., Int J Pharm 366: 218-220 (2009); Adjei andGarren, Pharmaceutical Research, 7(6): 565-569 (1990); Kawashima et al.,J Controlled Release 62(1-2): 279-287 (1999); Liu et al., Pharm Res10(2): 228-232 (1993); International Patent Application Publication Nos.WO 2007/133747 and WO 2007/141411.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous. The analog of the present disclosure can be administeredwith a physiologically acceptable diluent in a pharmaceutical carrier,such as a sterile liquid or mixture of liquids, including water, saline,aqueous dextrose and related sugar solutions, an alcohol, such asethanol or hexadecyl alcohol, a glycol, such as propylene glycol orpolyethylene glycol, dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-153-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind known in the art.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)).

Additionally, the analog of the present disclosures can be made intosuppositories for rectal administration by mixing with a variety ofbases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the analog of thedisclosure can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes.

Dose

The analogs of the disclosure are believed to be useful in methods oftreating a disease or medical condition in which glucagon receptoragonism, GLP-1 receptor agonism, or Glucagon receptor/GLP-1 receptorco-agonism plays a role. For purposes of the disclosure, the amount ordose of the analog of the present disclosure administered should besufficient to effect, e.g., a therapeutic or prophylactic response, inthe subject or animal over a reasonable time frame. For example, thedose of the analog of the present disclosure should be sufficient tostimulate cAMP secretion from cells as described herein or sufficient todecrease blood glucose levels, fat levels, food intake levels, or bodyweight of a mammal, in a period of from about 1 to 4 minutes, 1 to 4hours or 1 to 4 weeks or longer, e.g., 5 to 20 or more weeks, from thetime of administration. In certain embodiments, the time period could beeven longer. The dose will be determined by the efficacy of theparticular analog of the present disclosure and the condition of theanimal (e.g., human), as well as the body weight of the animal (e.g.,human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes herein, an assay, which comprises comparing the extent towhich blood glucose levels are lowered upon administration of a givendose of the analog of the present disclosure to a mammal among a set ofmammals of which is each given a different dose of the analog, could beused to determine a starting dose to be administered to a mammal. Theextent to which blood glucose levels are lowered upon administration ofa certain dose can be assayed by methods known in the art, including,for instance, the methods described herein as Example 4.

The dose of the analog of the present disclosure also will be determinedby the existence, nature and extent of any adverse side effects thatmight accompany the administration of a particular analog of the presentdisclosure. Typically, the attending physician will decide the dosage ofthe analog of the present disclosure with which to treat each individualpatient, taking into consideration a variety of factors, such as age,body weight, general health, diet, sex, analog of the present disclosureto be administered, route of administration, and the severity of thecondition being treated. By way of example and not intending to limitthe invention, the dose of the analog of the present disclosure can beabout 0.0001 to about 1 g/kg body weight of the subject beingtreated/day, from about 0.0001 to about 0.001 g/kg body weight/day, orabout 0.01 mg to about 1 g/kg body weight/day.

In some embodiments, the pharmaceutical composition comprises any of theanalogs disclosed herein at a purity level suitable for administrationto a patient. In some embodiments, the analog has a purity level of atleast about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98% or about 99%, and a pharmaceuticallyacceptable diluent, carrier or excipient. The pharmaceutical compositionin some aspects comprise the analog of the present disclosure at aconcentration of at least A, wherein A is about 0.001 mg/ml, about 0.01mg/ml, 0 about 1 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml,about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg/ml,about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml,about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25mg/ml or higher. In some embodiments, the pharmaceutical compositioncomprises the analog at a concentration of at most B, wherein B is about30 mg/ml, about 25 mg/ml, about 24 mg/ml, about 23, mg/ml, about 22mg/ml, about 21 mg/ml, about 20 mg/ml, about 19 mg/ml, about 18 mg/ml,about 17 mg/ml, about 16 mg/ml, about 15 mg/ml, about 14 mg/ml, about 13mg/ml, about 12 mg/ml, about 11 mg/ml, about 10 mg/ml, about 9 mg/ml,about 8 mg/ml, about 7 mg/ml, about 6 mg/ml, about 5 mg/ml, about 4mg/ml, about 3 mg/ml, about 2 mg/ml, about 1 mg/ml, or about 0.1 mg/ml.In some embodiments, the compositions may contain an analog at aconcentration range of A to B mg/ml, for example, about 0.001 to about30.0 mg/ml.

Targeted Forms

One of ordinary skill in the art will readily appreciate that theanalogs of the disclosure can be modified in any number of ways, suchthat the therapeutic or prophylactic efficacy of the analog of thepresent disclosures is increased through the modification. For instance,the analog of the present disclosure can be conjugated either directlyor indirectly through a linker to a targeting moiety. The practice ofconjugating compounds, e.g., glucagon analogs described herein, totargeting moieties is known in the art. See, for instance, Wadhwa etal., J Drug Targeting, 3, 111-127 (1995) and U.S. Pat. No. 5,087,616.The term “targeting moiety” as used herein, refers to any molecule oragent that specifically recognizes and binds to a cell-surface receptor,such that the targeting moiety directs the delivery of the analog of thepresent disclosures to a population of cells on which surface thereceptor (the glucagon receptor, the GLP-1 receptor) is expressed.Targeting moieties include, but are not limited to, antibodies, orfragments thereof, peptides, hormones, growth factors, cytokines, andany other natural or non-natural ligands, which bind to cell surfacereceptors (e.g., Epithelial Growth Factor Receptor (EGFR), T-cellreceptor (TCR), B-cell receptor (BCR), CD28, Platelet-derived GrowthFactor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).As used herein a “linker” is a bond, molecule or group of molecules thatbinds two separate entities to one another. Linkers may provide foroptimal spacing of the two entities or may further supply a labilelinkage that allows the two entities to be separated from each other.Labile linkages include photocleavable groups, acid-labile moieties,base-labile moieties and enzyme-cleavable groups. The term “linker” insome embodiments refers to any agent or molecule that bridges the analogof the present disclosures to the targeting moiety. One of ordinaryskill in the art recognizes that sites on the analog of the presentdisclosures, which are not necessary for the function of the analog ofthe present disclosures, are ideal sites for attaching a linker and/or atargeting moiety, provided that the linker and/or targeting moiety, onceattached to the analog of the present disclosures, do(es) not interferewith the function of the analog of the present disclosures, i.e., theability to stimulate cAMP secretion from cells, to treat diabetes orobesity.

Controlled Release Formulations

Alternatively, the glucagon analogs described herein can be modifiedinto a depot form, such that the manner in which the analog of thepresent disclosures is released into the body to which it isadministered is controlled with respect to time and location within thebody (see, for example, U.S. Pat. No. 4,450,150). Depot forms of analogof the present disclosures can be, for example, an implantablecomposition comprising the analog of the present disclosures and aporous or non-porous material, such as a polymer, wherein the analog ofthe present disclosures is encapsulated by or diffused throughout thematerial and/or degradation of the non-porous material. The depot isthen implanted into the desired location within the body and the analogof the present disclosures are released from the implant at apredetermined rate.

The pharmaceutical composition in certain aspects is modified to haveany type of in vivo release profile. In some aspects, the pharmaceuticalcomposition is an immediate release, controlled release, sustainedrelease, extended release, delayed release, or bi-phasic releaseformulation. Methods of formulating peptides for controlled release areknown in the art. See, for example, Qian et al., J Pharm 374: 46-52(2009) and International Patent Application Publication Nos. WO2008/130158, WO2004/033036; WO2000/032218; and WO 1999/040942.

The instant compositions may further comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. The disclosed pharmaceutical formulations may be administeredaccording to any regime including, for example, daily (1 time per day, 2times per day, 3 times per day, 4 times per day, 5 times per day, 6times per day), every two days, every three days, every four days, everyfive days, every six days, weekly, bi-weekly, every three weeks,monthly, or bi-monthly.

Combinations

The glucagon analogs described herein may be administered alone or incombination with other therapeutic agents which aim to treat or preventany of the diseases or medical conditions described herein. For example,the glucagon analogs described herein may be co-administered with(simultaneously or sequentially) an anti-diabetic or anti-obesity agent.Anti-diabetic agents known in the art or under investigation includeinsulin, leptin, Peptide YY (PYY), Pancreatic Peptide (PP), fibroblastgrowth factor 21 (FGF21), Y2Y4 receptor agonists, sulfonylureas, such astolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase),chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta,Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron);meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix);biguanides such as metformin (Glucophage) or phenformin;thiazolidinediones such as rosiglitazone (Avandia), pioglitazone(Actos), or troglitazone (Rezulin), or other PPARγ inhibitors; alphaglucosidase inhibitors that inhibit carbohydrate digestion, such asmiglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) orpramlintide; Dipeptidyl peptidase-4 (DPP-4) inhibitors such asvildagliptin or sitagliptin; SGLT (sodium-dependent glucosetransporter 1) inhibitors; glucokinase activators (GKA); glucagonreceptor antagonists (GRA); or FBPase (fructose 1,6-bisphosphatase)inhibitors.

Anti-obesity agents known in the art or under investigation includeappetite suppressants, including phenethylamine type stimulants,phentermine (optionally with fenfluramine or dexfenfluramine),diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®),benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant(Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin;fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine),Excalia (bupropion and zonisamide) or Contrave (bupropion andnaltrexone); or lipase inhibitors, similar to XENICAL (Orlistat) orCetilistat (also known as ATL-962), or GT 389-255.

The peptides described herein in some embodiments are co-administeredwith an agent for treatment of non-alcoholic fatty liver disease orNASH. Agents used to treat non-alcoholic fatty liver disease includeursodeoxycholic acid (a.k.a., Actigall, URSO, and Ursodiol), Metformin(Glucophage), rosiglitazone (Avandia), Clofibrate, Gemfibrozil,Polymixin B, and Betaine.

The peptides described herein in some embodiments are co-administeredwith an agent for treatment of a neurodegenerative disease, e.g.,Parkinson's Disease. Anti-Parkinson's Disease agents are furthermoreknown in the art and include, but not limited to, levodopa, carbidopa,anticholinergics, bromocriptine, pramipexole, and ropinirole,amantadine, and rasagiline.

In view of the foregoing, the invention further provides pharmaceuticalcompositions and kits additionally comprising one of these othertherapeutic agents. The additional therapeutic agent may be administeredsimultaneously or sequentially with the analog of the presentdisclosure. In some aspects, the analog is administered before theadditional therapeutic agent, while in other aspects, the analog isadministered after the additional therapeutic agent.

Uses

It is contemplated that the glucagon analogs described herein andrelated pharmaceutical compositions are useful for treatment of adisease or medical condition, in which e.g., the lack of activity at theglucagon receptor, the GLP-1 receptor, or at both receptors, is a factorin the onset and/or progression of the disease or medical condition.Accordingly, the invention provides a method of treating or preventing adisease or medical condition in a patient, wherein the disease ormedical condition is a disease of medical condition in which a lack ofGLP-1 receptor activation and/or glucagon receptor activation isassociated with the onset and/or progression of the disease of medicalcondition. The method comprises providing to the patient an analog inaccordance with any of those described herein in an amount effective totreat or prevent the disease or medical condition.

In some embodiments, the disease or medical condition is metabolicsyndrome. Metabolic Syndrome, also known as metabolic syndrome X,insulin resistance syndrome or Reaven's syndrome, is a disorder thataffects over 50 million Americans. Metabolic Syndrome is typicallycharacterized by a clustering of at least three or more of the followingrisk factors: (1) abdominal obesity (excessive fat tissue in and aroundthe abdomen), (2) atherogenic dyslipidemia (blood fat disordersincluding high triglycerides, low HDL cholesterol and high LDLcholesterol that enhance the accumulation of plaque in the arterywalls), (3) elevated blood pressure, (4) insulin resistance or glucoseintolerance, (5) prothrombotic state (e.g., high fibrinogen orplasminogen activator inhibitor-1 in blood), and (6) pro-inflammatorystate (e.g., elevated C-reactive protein in blood). Other risk factorsmay include aging, hormonal imbalance and genetic predisposition.

Metabolic Syndrome is associated with an increased the risk of coronaryheart disease and other disorders related to the accumulation ofvascular plaque, such as stroke and peripheral vascular disease,referred to as atherosclerotic cardiovascular disease (ASCVD). Patientswith Metabolic Syndrome may progress from an insulin resistant state inits early stages to full blown type II diabetes with further increasingrisk of ASCVD. Without intending to be bound by any particular theory,the relationship between insulin resistance, Metabolic Syndrome andvascular disease may involve one or more concurrent pathogenicmechanisms including impaired insulin-stimulated vasodilation, insulinresistance-associated reduction in NO availability due to enhancedoxidative stress, and abnormalities in adipocyte-derived hormones suchas adiponectin (Lteif and Mather, Can. J. Cardiol. 20 (suppl. B):66B-76B(2004)).

According to the 2001 National Cholesterol Education Program AdultTreatment Panel (ATP III), any three of the following traits in the sameindividual meet the criteria for Metabolic Syndrome: (a) abdominalobesity (a waist circumference over 102 cm in men and over 88 cm inwomen); (b) serum triglycerides (150 mg/dl or above); (c) HDLcholesterol (40 mg/dl or lower in men and 50 mg/dl or lower in women);(d) blood pressure (130/85 or more); and (e) fasting blood glucose (110mg/dl or above). According to the World Health Organization (WHO), anindividual having high insulin levels (an elevated fasting blood glucoseor an elevated post meal glucose alone) with at least two of thefollowing criteria meets the criteria for Metabolic Syndrome: (a)abdominal obesity (waist to hip ratio of greater than 0.9, a body massindex of at least 30 kg/m2, or a waist measurement over 37 inches); (b)cholesterol panel showing a triglyceride level of at least 150 mg/dl oran HDL cholesterol lower than 35 mg/dl; (c) blood pressure of 140/90 ormore, or on treatment for high blood pressure). (Mathur, Ruchi,“Metabolic Syndrome,” ed. Shiel, Jr., William C., MedicineNet.com, May11, 2009).

For purposes herein, if an individual meets the criteria of either orboth of the criteria set forth by the 2001 National CholesterolEducation Program Adult Treatment Panel or the WHO, that individual isconsidered as afflicted with Metabolic Syndrome.

Without being bound to any particular theory, peptides described hereinare useful for treating Metabolic Syndrome. Accordingly, the inventionprovides a method of preventing or treating Metabolic Syndrome, orreducing one, two, three or more risk factors thereof, in a subject,comprising providing to the subject an analog described herein in anamount effective to prevent or treat Metabolic Syndrome, or the riskfactor thereof.

In some embodiments, the method treats a hyperglycemic medicalcondition. In certain aspects, the hyperglycemic medical condition isdiabetes, diabetes mellitus type I, diabetes mellitus type II, orgestational diabetes, either insulin-dependent or non-insulin-dependent.In some aspects, the method treats the hyperglycemic medical conditionby reducing one or more complications of diabetes including nephropathy,retinopathy and vascular disease.

In some aspects, the disease or medical condition is obesity. In someaspects, the obesity is drug-induced obesity. In some aspects, themethod treats obesity by preventing or reducing weight gain orincreasing weight loss in the patient. In some aspects, the methodtreats obesity by reducing appetite, decreasing food intake, loweringthe levels of fat in the patient, or decreasing the rate of movement offood through the gastrointestinal system.

Because obesity is associated with the onset or progression of otherdiseases, the methods of treating obesity are further useful in methodsof reducing complications associated with obesity including vasculardisease (coronary artery disease, stroke, peripheral vascular disease,ischemia reperfusion, etc.), hypertension, onset of diabetes type II,hyperlipidemia and musculoskeletal diseases. The invention accordinglyprovides methods of treating or preventing these obesity-associatedcomplications.

In some embodiments, the disease or medical condition is Nonalcoholicfatty liver disease (NAFLD). NAFLD refers to a wide spectrum of liverdisease ranging from simple fatty liver (steatosis), to nonalcoholicsteatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring ofthe liver). All of the stages of NAFLD have in common the accumulationof fat (fatty infiltration) in the liver cells (hepatocytes). Simplefatty liver is the abnormal accumulation of a certain type of fat,triglyceride, in the liver cells with no inflammation or scarring. InNASH, the fat accumulation is associated with varying degrees ofinflammation (hepatitis) and scarring (fibrosis) of the liver. Theinflammatory cells can destroy the liver cells (hepatocellularnecrosis). In the terms “steatohepatitis” and “steatonecrosis”, steatorefers to fatty infiltration, hepatitis refers to inflammation in theliver, and necrosis refers to destroyed liver cells. NASH can ultimatelylead to scarring of the liver (fibrosis) and then irreversible, advancedscarring (cirrhosis). Cirrhosis that is caused by NASH is the last andmost severe stage in the NAFLD spectrum. (Mendler, Michel, “Fatty Liver:Nonalcoholic Fatty Liver Disease (NAFLD) and NonalcoholicSteatohepatitis (NASH),” ed. Schoenfield, Leslie J., MedicineNet.com,Aug. 29, 2005).

Alcoholic Liver Disease, or Alcohol-Induced Liver Disease, encompassesthree pathologically distinct liver diseases related to or caused by theexcessive consumption of alcohol: fatty liver (steatosis), chronic oracute hepatitis, and cirrhosis. Alcoholic hepatitis can range from amild hepatitis, with abnormal laboratory tests being the only indicationof disease, to severe liver dysfunction with complications such asjaundice (yellow skin caused by bilirubin retention), hepaticencephalopathy (neurological dysfunction caused by liver failure),ascites (fluid accumulation in the abdomen), bleeding esophageal varices(varicose veins in the esophagus), abnormal blood clotting and coma.Histologically, alcoholic hepatitis has a characteristic appearance withballooning degeneration of hepatocytes, inflammation with neutrophilsand sometimes Mallory bodies (abnormal aggregations of cellularintermediate filament proteins). Cirrhosis is characterized anatomicallyby widespread nodules in the liver combined with fibrosis. (Worman,Howard J., “Alcoholic Liver Disease”, Columbia University Medical Centerwebsite).

Without being bound to any particular theory, the analogs describedherein are useful for the treatment of Alcoholic Liver Disease, NAFLD,or any stage thereof, including, for example, steatosis,steatohepatitis, hepatitis, hepatic inflammation, NASH, cirrhosis, orcomplications thereof. Accordingly, the invention provides a method ofpreventing or treating Alcoholic Liver Disease, NAFLD, or any stagethereof, in a subject comprising providing to a subject an analogdescribed herein in an amount effective to prevent or treat AlcoholicLiver Disease, NAFLD, or the stage thereof. Such treatment methodsinclude reduction in one, two, three or more of the following: liver fatcontent, incidence or progression of cirrhosis, incidence ofhepatocellular carcinoma, signs of inflammation, e.g., abnormal hepaticenzyme levels (e.g., aspartate aminotransferase AST and/or alanineaminotransferase ALT, or LDH), elevated serum ferritin, elevated serumbilirubin, and/or signs of fibrosis, e.g., elevated TGF-beta levels. Inpreferred embodiments, the peptides are used treat patients who haveprogressed beyond simple fatty liver (steatosis) and exhibit signs ofinflammation or hepatitis. Such methods may result, for example, inreduction of AST and/or ALT levels.

GLP-1 and exendin-4 have been shown to have some neuroprotective effect.The invention also provides uses of the glucagon analogs describedherein in treating neurodegenerative diseases, including but not limitedto Alzheimer's disease, Parkinson's disease, Multiple Sclerosis,Amylotrophic Lateral Sclerosis, other demyelination related disorders,senile dementia, subcortical dementia, arteriosclerotic dementia,AIDS-associated dementia, or other dementias, a central nervous systemcancer, traumatic brain injury, spinal cord injury, stroke or cerebralischemia, cerebral vasculitis, epilepsy, Huntington's disease,Tourette's syndrome, Guillain Barre syndrome, Wilson disease, Pick'sdisease, neuroinflammatory disorders, encephalitis, encephalomyelitis ormeningitis of viral, fungal or bacterial origin, or other centralnervous system infections, prion diseases, cerebellar ataxias,cerebellar degeneration, spinocerebellar degeneration syndromes,Friedreichs ataxia, ataxia telangiectasia, spinal dysmyotrophy,progressive supranuclear palsy, dystonia, muscle spasticity, tremor,retinitis pigmentosa, striatonigral degeneration, mitochondrialencephalo-myopathies, neuronal ceroid lipofuscinosis, hepaticencephalopathies, renal encephalopathies, metabolic encephalopathies,toxin-induced encephalopathies, and radiation-induced brain damage.

In some embodiments, the disease or medical condition is hypoglycemia.In some embodiments, the patient is a diabetic patient and thehypoglycemia is induced by the administration of insulin. In specificaspects, the method comprises providing the analog of the presentdisclosure in combination with insulin so that the analog buffers thehypoglycemic effects of the bolus administration of insulin.

In some embodiments, the glucagon analogs are used in conjunction withparenteral administration of nutrients to non-diabetic patients in ahospital setting, e.g., to patients receiving parenteral nutrition ortotal parenteral nutrition. Nonlimiting examples include surgerypatients, patients in comas, patients with digestive tract illness, or anonfunctional gastrointestinal tract (e.g. due to surgical removal,blockage or impaired absorptive capacity, Crohn's disease, ulcerativecolitis, gastrointestinal tract obstruction, gastrointestinal tractfistula, acute pancreatitis, ischemic bowel, major gastrointestinalsurgery, certain congenital gastrointestinal tract anomalies, prolongeddiarrhea, or short bowel syndrome due to surgery, patients in shock, andpatients undergoing healing processes often receive parenteraladministration of carbohydrates along with various combinations oflipids, electrolytes, minerals, vitamins and amino acids. The glucagonanalogs and the parenteral nutrition composition can be administered atthe same time, at different times, before, or after each other, providedthat the glucagon analog is exerting the desired biological effect atthe time that the parenteral nutrition composition is being digested.For example, the parenteral nutrition may be administered, 1, 2 or 3times per day, while the glucagon analog is administered once everyother day, three times a week, two times a week, once a week, once every2 weeks, once every 3 weeks, or once a month.

As used herein, the terms “treat,” and “prevent” as well as wordsstemming therefrom, do not necessarily imply 100% or complete treatmentor prevention. Rather, there are varying degrees of treatment orprevention of which one of ordinary skill hi the art recognizes ashaving a potential benefit or therapeutic effect. In this respect, theinventive methods can provide any amount of any level of treatment orprevention of a disease or medical condition in a mammal. Furthermore,the treatment or prevention provided by the method can include treatmentor prevention of one or more conditions or symptoms of the disease ormedical condition. For example, with regard to methods of treatingobesity, the method in some embodiments, achieves a decrease in foodintake by or fat levels in a patient. Also, for purposes herein,“prevention” can encompass delaying the onset of the disease, or asymptom or condition thereof.

With regard to the above methods of treatment, the patient is any host.In some embodiments, the host is a mammal. As used herein, the term“mammal” refers to any vertebrate animal of the mammalia class,including, but not limited to, any of the monotreme, marsupial, andplacental taxas. In some embodiments, the mammal is one of the mammalsof the order Rodentia, such as mice and hamsters, and mammals of theorder Logomorpha, such as rabbits. In certain embodiments, the mammalsare from the order Carnivora, including Felines (cats) and Canines(dogs). In certain embodiments, the mammals are from the orderArtiodactyla, including Bovines (cows) and Swines (pigs) or of the orderPerssodactyla, including Equines (horses). In some instances, themammals are of the order Primates, Ceboids, or Simoids (monkeys) or ofthe order Anthropoids (humans and apes). In particular embodiments, themammal is a human.

Kits

The glucagon analogs of the present disclosure can be provided inaccordance with one embodiment as part of a kit. Accordingly, in someembodiments, a kit for administering a glucagon analog, e.g., a glucagonagonist peptide, to a patient in need thereof is provided wherein thekit comprises a glucagon analog as described herein.

In one embodiment the kit is provided with a device for administeringthe glucagon composition to a patient, e.g., syringe needle, pen device,jet injector or other needle-free injector. The kit may alternatively orin addition include one or more containers, e.g., vials, tubes, bottles,single or multi-chambered pre-filled syringes, cartridges, infusionpumps (external or implantable), jet injectors, pre-filled pen devicesand the like, optionally containing the glucagon analog in a lyophilizedform or in an aqueous solution. The kits in some embodiments compriseinstructions for use. In accordance with one embodiment the device ofthe kit is an aerosol dispensing device, wherein the composition isprepackaged within the aerosol device. In another embodiment the kitcomprises a syringe and a needle, and in one embodiment the sterileglucagon composition is prepackaged within the syringe.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1 Synthesis of Peptide Fragments of Glucagon

Materials:

All peptides described herein in the EXAMPLES were amidated unlessspecified otherwise.

MBHA resin (4-methylbenzhydrylamine polystyrene resin was used duringpeptide synthesis. MBHA resin, 100-180 mesh, 1% DVB cross-linkedpolystyrene; loading of 0.7-1.0 mmol/g), Boc-protected and Fmocprotected amino acids were purchased from Midwest Biotech. The solidphase peptide syntheses using Boc-protected amino acids were performedon an Applied Biosystem 430A Peptide Synthesizer. Fmoc protected aminoacid synthesis was performed using the Applied Biosystems Model 433Peptide Synthesizer.

Peptide Synthesis (Boc Amino Acids/HF Cleavage):

Synthesis of these analogs was performed on the Applied Biosystem Model430A Peptide Synthesizer. Synthetic peptides were constructed bysequential addition of amino acids to a cartridge containing 2 mmol ofBoc protected amino acid. Specifically, the synthesis was carried outusing Boc DEPBT-activated single couplings. At the end of the couplingstep, the peptidyl-resin was treated with TFA to remove the N-terminalBoc protecting group. It was washed repeatedly with DMF and thisrepetitive cycle was repeated for the desired number of coupling steps.After the assembly, the sidechain protection, Fmoc, was removed by 20%piperidine treatment and acylation was conducted using DIC. Thepeptidyl-resin at the end of the entire synthesis was dried by usingDCM, and the peptide was cleaved from the resin with anhydrous HF.

For the lactamization, orthogonal protecting groups were selected forGlu and Lys (e.g., Glu(Fm), Lys(Fmoc)). After removal of the protectinggroups and before HF cleavage, cyclization was performed as describedpreviously (see, e.g., International Patent Application Publication No.WO2008/101017).

HF Treatment of the Peptidyl-Resin

The peptidyl-resin was treated with anhydrous HF, and this typicallyyielded approximately 350 mg (˜50% yield) of a crudedeprotected-peptide. Specifically, the peptidyl-resin (30 mg to 200 mg)was placed in the hydrogen fluoride (HF) reaction vessel for cleavage.500 μL of p-cresol was added to the vessel as a carbonium ion scavenger.The vessel was attached to the HF system and submerged in themethanol/dry ice mixture. The vessel was evacuated with a vacuum pumpand 10 ml of HF was distilled to the reaction vessel. This reactionmixture of the peptidyl-resin and the HF was stirred for one hour at 0°C., after which a vacuum was established and the HF was quicklyevacuated (10-15 min). The vessel was removed carefully and filled withapproximately 35 ml of ether to precipitate the peptide and to extractthe p-cresol and small molecule organic protecting groups resulting fromHF treatment. This mixture was filtered utilizing a teflon filter andrepeated twice to remove all excess cresol. This filtrate was discarded.The precipitated peptide dissolves in approximately 20 ml of 10% aceticacid (aq). This filtrate, which contained the desired peptide, wascollected and lyophilized.

An analytical HPLC analysis of the crude solubilized peptide wasconducted under the following conditions [4.6×30 mm Xterra C8, 1.50mL/min, 220 nm, A buffer 0.1% TFA/10% ACN, B buffer 0.1% TFA/100% ACN,gradient 5-95% B over 15 minutes]. The extract was diluted twofold withwater and loaded onto a 2.2×25 cm Vydac C4 preparative reverse phasecolumn and eluted using an acetonitrile gradient on a Waters HPLC system(A buffer of 0.1% TFA/10% ACN, B buffer of 0.1% TFA/10% CAN and agradient of 0-100% B over 120 minutes at a flow of 15.00 ml/min. HPLCanalysis of the purified peptide demonstrated greater than 95% purityand electrospray ionization mass spectral analysis was used to confirmthe identity of the peptide.

Peptide Acylation

Acylated peptides were prepared as follows. Peptides were synthesized ona solid support resin using either a CS Bio 4886 Peptide Synthesizer orApplied Biosystems 430A Peptide Synthesizer. In situ neutralizationchemistry was used as described by Schnolzer et al., Int. J. PeptideProtein Res. 40: 180-193 (1992). For acylated peptides, the target aminoacid residue to be acylated (e.g., position ten, relative to the aminoacid position numbering of SEQ ID NO: 3) was substituted with an Nε-FMOC lysine residue. Treatment of the completed N-terminally BOCprotected peptide with 20% piperidine in DMF for 30 minutes removedFMOC/formyl groups. Coupling to the free ε-amino Lys residue wasachieved by coupling a ten-fold molar excess of either an FMOC-protectedspacer amino acid (ex. FMOC-Glu-OtBu) or acyl chain (ex.CH₃(CH₂)₁₄—COOH) and PyBOP or DEPBT coupling reagent in DMF/DIEA.Subsequent removal of the spacer amino acid's FMOC group is followed byrepetition of coupling with an acyl chain. Final treatment with 100% TFAresulted in removal of any side chain protecting groups and theN-terminal BOC group. Peptide resins were neutralized with 5% DIEA/DMF,dried, and then cleaved from the support using HF/p-cresol, 95:5, at 0°C. for one hour. Following ether extraction, a 5% HOAc solution was usedto solvate the crude peptide. A sample of the solution was then verifiedto contain the correct molecular weight peptide by ESI-MS. Correctpeptides were purified by RP-HPLC using a linear gradient of 10%CH3CN/0.1% TFA to 0.1% TFA in 100% CH3CN. A Vydac C18 22 mm×250 mmprotein column was used for the purification. Acylated peptide analogsgenerally completed elution by a buffer ratio of 20:80. Portions werepooled together and checked for purity on an analytical RP-HPLC. Purefractions were lyophilized yielding white, solid peptides.

If a peptide comprised a lactam bridge and target residues to beacylated, acylation is carried out as described above upon addition ofthat amino acid to the peptide backbone.

Peptide PEGylation

For peptide PEGylation, 40 kDa methoxy poly(ethylene glycol)idoacetamide (NOF) was reacted with a molar equivalent of peptide in 7MUrea, 50 mM Tris-HCl buffer using the minimal amount of solvent neededto dissolve both peptide and PEG into a clear solution (generally lessthan 2 mL for a reaction using 2-3 mg peptide). Vigorous stirring atroom temperature commenced for 4-6 hours and the reaction analyzed byanalytical RP-HPLC. PEGylated products appeared distinctly from thestarting material with decreased retention times. Purification wasperformed on a Vydac C4 column with conditions similar to those used forthe initial peptide purification. Elution occurred around buffer ratiosof 50:50. Fractions of pure PEGylated peptide were found andlyophilized. Yields were above 50%, varying per reaction.

Analysis Using Mass Spectrometry

The mass spectra were obtained using a Sciex API-III electrosprayquadrapole mass spectrometer with a standard ESI ion source. Ionizationconditions that were used are as follows: ESI in the positive-ion mode;ion spray voltage, 3.9 kV; orifice potential, 60 V. The nebulizing andcurtain gas used was nitrogen flow rate of 0.9 L/min. Mass spectra wererecorded from 600-1800 Thompsons at 0.5 Th per step and 2 msec dwelltime. The sample (about 1 mg/mL) was dissolved in 50% aqueousacetonitrile with 1% acetic acid and introduced by an external syringepump at the rate of 5 μL/min.

When the peptides were analyzed in PBS solution by ESI MS, they werefirst desalted using a ZipTip solid phase extraction tip containing 0.6μL C4 resin, according to instructions provided by the manufacturer(Millipore Corporation, Billerica, Mass., see the Millipore website ofthe world wide web at millipore.com/catalogue.nsf/docs/C5737).

High Performance Liquid Chromatography (HPLC) Analysis:

Preliminary Analyses were Performed with these Crude Peptides to Get anapproximation of their relative conversion rates in Phosphate BufferedSaline (PBS) buffer (pH, 7.2) using high performance liquidchromatography (HPLC) and MALDI analysis. The crude peptide samples weredissolved in the PBS buffer at a concentration of 1 mg/ml. 1 ml of theresulting solution was stored in a 1.5 ml HPLC vial which was thensealed and incubated at 37° C. Aliquots of 100 μl were drawn out atvarious time intervals, cooled to room temperature and analyzed by HPLC.

The HPLC analyses were performed using a Beckman System GoldChromatography system using a UV detector at 214 nm. HPLC analyses wereperformed on a 150 mm×4.6 mm C18 Vydac column. The flow rate was 1ml/min. Solvent A contained 0.1% TFA in distilled water, and solvent Bcontained 0.1% TFA in 90% CH3CN. A linear gradient was employed (40% to70% B in 15 minutes). The data were collected and analyzed using PeakSimple Chromatography software.

The initial rates of hydrolysis were used to measure the rate constantfor the dissociation of the respective prodrugs. The concentrations ofthe prodrug and the drug were estimated from their peak areasrespectively. The first order dissociation rate constants of theprodrugs were determined by plotting the logarithm of the concentrationof the prodrug at various time intervals. The slope of this plot givesthe rate constant ‘k’. The half lives of the degradation of the variousprodrugs were then calculated by using the formula t½=0.693/k.

Example 2 Synthesis of the Peptide of Seq ID No. 17

The peptide seq ID no. 17 was synthesized by solid phase using Fmoc/t-Buchemistry. For the preparation of the peptide, 0.5 g of2-Chlorotrityl-resin (100-200 mesh, 1.18 mmol/g) (GL Biochem) were firstderivatized by incubation with 0.8 equivalents (eq.). Fmoc-Gln(Trt)-OH,4 eq. relative to amino acid diisopropylethylamine (DIPEA) in dry DCM(dichloromethane) for 2 hours. The peptide sequence assembly was thenperformed on a peptide multisynthesizer APEX396 (Advanced Biotech). Allthe amino acids were dissolved at a 0.5M concentration in DMF(N,N-dimethyl formamide). The acylation reactions were performed for 60min with 6-fold excess of activated amino acid over the resin free aminogroups. Double acylations were performed for Aib2, Aib16, Asp15, His1.The amino acids were activated with equimolar amounts of HATU(2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate), solution 0.5 M in DMF, and a 2-fold molar excessof DIEA (N,N-diisopropylethylamine), solution 2 M in NMP(1-methyl-2-pyrrolidinone).

The side chain protecting groups were: tert-butyl for Asp and Glu, Ser,Thr and Tyr; trityl for Asn, Gln and His; tert-butoxy-carbonyl for Lys,Trp; and, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for Arg;Boc-His(Trt)-OH was used in the synthesis.

The lysine at position 10, to be derivatized on the side chain, wasincorporated as Lys(Alloc). At the end of the assembly the Allocprotecting group was removed and the synthesis was completed bycondensation of the two γ-carboxyglutamic acid residues (4 eq.) and thepalmitic acid (4 eq.) using HBTU (4 eq.;(O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate),and DIPEA (8 eq.) as activators.

At the end of the synthesis, the dry peptide-resins were individuallytreated with 25 mL of the cleavage mixture, 88% trifluoroacetic acid(TFA), 5% phenol, 2% triisopropylsilane and 5% water for 2 hours at roomtemperature. The resin was filtered and the volume of the solution wasreduced then added to cold methyl-t-butyl ether in order to precipitatethe peptide. After centrifugation, the peptide pellets were washed withfresh cold methyl-t-butyl ether to remove the organic scavengers. Theprocess was repeated twice. Final pellets were dried, resuspended inH₂O, 20% acetonitrile, and lyophilized.

The crude peptides were purified by reverse-phase HPLC using preparativeWaters XBridge C18 (50×150 mm, 5 μm) and using as eluents (A) 0.1% TFAin water and (B) 0.1% TFA in acetonitrile. Analytical UPLC was performedon a Waters Chromatograph, with a BEH130, C18 Acquity, 1.7 μm column,2.1×100 mm, (Waters), at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1%TFA (B) as solvents. The purified peptide was characterized byelectrospray mass spectrometry on a Waters SQ Detector.

Example 3

The ability of each peptide to induce cAMP was measured in a fireflyluciferase-based reporter assay. The cAMP production that is induced isdirectly proportional to the glucagon fragment binding to the glucagonor GLP-1 receptor. HEK293 cells co-transfected with the glucagon orGLP-1 receptor, respectively, and luciferase gene linked to a cAMPresponsive element were employed for the bioassay.

The cells were serum-deprived by culturing 16 hours in Dulbecco-modifiedMinimum Essential Medium (Invitrogen, Carlsbad, Calif.) supplementedwith 0.25% Bovine Growth Serum (HyClone, Logan, Utah) and then incubatedwith serial dilutions of glucagon fragments for 5 hours at 37° C., 5%CO2 in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences,San Jose, Calif.). At the end of the incubation, 100 μL of LucLiteluminescence substrate reagent (Perkin Elmer, Wellesley, Mass.) wereadded to each well. The plate was shaken briefly, incubated 10 min inthe dark and light output was measured on MicroBeta-1450 liquidscintillation counter (Perkin-Elmer, Wellesley, Mass.). The effective50% concentrations (EC50) were calculated by using Origin software(OriginLab, Northampton, Mass.).

Example 4

The in vitro agonist potencies of the peptide of SEQ ID NO. 17 weredetermined using Chinese Hamster Ovary cells (CHO-K1) stably expressinghuman GLP-1R, GCGR or GIPR. The GLP-1R and GCGR stable cell lineisogenic pools were generated by Invitrogen™ using their Jump-In™targeted integration technology, whereas the stable CHO/GIPR lines weregenerated through classic cloning and transfection techniques (randomintegration) with limited dilution for selection of clones. The peptideswere tested in vitro for their relative abilities to stimulate cAMPproduction in the various cell lines using the DiscoveRx Hithunter™ cAMPXS+ assay kit (cat#90-0075L) as per kit instructions. The peptide 50%effective concentrations (EC50s) were calculated by non-linearregression analysis 4-parameter curve fitting with GraphPad Prism 4software (GraphPad Software, San Diego, Calif.) by plotting luminescencevalues versus peptide doses.

Example 5

Peptides having the amino acid sequences as described in the sequencelisting were made as essentially described in Example 1 or 2 andsubsequently tested for in vitro agonist activity at each of theglucagon receptor and GLP-1 receptor as essentially described in Example3 or 4. In some instances, the in vitro agonist activity at the GIPreceptor was also tested. The results are shown below in Table 1.

TABLE 1 EC₅₀ EC₅₀ EC₅₀ SEQ (nM) at (nM) at (nM) at ID glucagon GLP-1 GIPNO: Sequence receptor receptor receptor 12 HaibQGTFTSDK(γGlu-γGlu- 0.0100.003 12.763 C16)SKYLDAibRAAQDFVQWLMDTKγGlu-amide 13HaibQGTFTSDYSKYLDERRA 2.806 0.567 139.639aibEFVC(40K-TE PEG)WLLDTKγE-amide 14 HsQGTFTSDK(γGlu-C16) 0.003 0.003976 SKYLDERAAQDFVQWLLDTKγGlu-amide 15 HaibQGTFTSDK(γGlu-γGlu-C16) 0.0060.005 1.901 SKYLDaibRAAQDFVQWLMornDTKQ-acid 16HaibQGTFTSDK(γGlu-γGlu-C16) 0.015 0.019 1.038SKYLDaibRAAQDFVQWLLDTKQ-acid 17 H aib QGTFTSD K(γGlu-γGlu- 0.110.075 >2000 C16)SKYLD aib RAAQDFVQWLMNTKγGlu-amide The EC50 values forthe peptides of SEQ ID Nos. 12-16 were obtained using the procedure ofExample 3. The EC50 value for the peptide of SEQ ID NO 17 was obtainedusing the procedure of Example 4.

As shown in Table 1, many of the peptides exhibit an EC50 at the GLP-1receptor in the nanomolar or picomolar range.

Example 6

Diet induced obesity (DIO) mice are divided into groups of eight miceper group and the initial average body weight of each group isdetermined. Each group of mice is subcutaneously injected daily with adose of a peptide or vehicle control for one week. The peptides testedin this study were the peptides of SEQ ID NOs: 12-16. The administereddoses varied between 1 and 10 nmol/kg for each of the peptides tested.Body weight, body composition, food intake, and blood glucose levelswere determined periodically throughout the test period.

To better determine the effect of these peptides on blood glucoselevels, a second experiment with db/db mice are performed. In thisexperiment, db/db mice are divided into groups of eight mice per groupand the initial average body weight of each group is determined. Eachgroup of mice is subcutaneously injected with a single dose of a peptideof one of SEQ ID NOs: 12-16, wherein the dose is within the range of 3and 30 nmole/kg. Body weight, body composition, food intake, and bloodglucose levels were determined periodically throughout the test period.

Example 7

The in vivo effects of certain peptides of the invention were confirmedin diet-induced obese (DIO) mice that were maintained on a high fat dietfor 16 weeks and had an initial body weight of ˜47 g. Mice wereadministered a vehicle control or a dose of a peptide daily for ninedays. The peptides tested in this study included a peptide of SEQ ID NO:12, a peptide of SEQ ID NO: 17, a peptide of SEQ ID NO: 18, and apeptide of SEQ ID NO: 19. Doses of each peptide were varied—each of thepeptides of SEQ ID NO: 12, 17, and 19 was administered at a dose ofeither 3 nmol/kg or 9 nmol/kg, while the peptide of SEQ ID NO: 18 wasadministered at a dose of 1 nmol/kg, 3 nmol/kg, or 9 nmol/kg. Cumulativebody weight change (in grams) was measured each day of the study and theresults are shown in FIG. 1. Data are expressed as mean±SEM.

Each of the peptides tested in this study demonstrated a body-weightlowering effect. Each group of mice that were administered a peptide ata 3 nmol/kg dose or a 9 nmol/kg dose demonstrated a decreased bodyweight (as compared to vehicle-treated mice) as early as Day 1 of thestudy. On Day 9 of the study, mice that were administered 3 nmol/kg ofthe peptide of SEQ ID NO: 12 exhibited an approximate 5.3% body weightreduction, while mice that were administered 9 nmol/kg dose of thepeptide of SEQ ID NO: 12 exhibited an approximate 29.8% body weightloss. On Day 9 of the study, mice that were administered 3 nmol/kg ofthe peptide of SEQ ID NO: 17 exhibited an approximate 9.6% body weightreduction, while mice that were administered 9 nmol/kg of the peptide ofSEQ ID NO: 17 demonstrated an approximate 35.5% weight loss. On Day 9 ofthe study, mice that were administered 3 nmol/kg of the peptide of SEQID NO: 18 exhibited an approximate 12.3% body weight reduction, whilemice that were administered 9 nmol/kg of the peptide of SEQ ID NO: 18demonstrated an approximate 26.8% body weight loss. On Day 9 of thisstudy, mice that were administered 3 nmol/kg of the peptide of SEQ IDNO: 19 demonstrated an approximate 8.1% body weight reduction, whereasmice that were administered 9 nmol/kg of the peptide of SEQ ID NO: 19exhibited an approximate 31.5% body weight loss.

In addition to cumulative body weight change, ambient glucose levels ofthe DIO mice were measured. As shown in FIG. 2, mice that wereadministered a 3 nmol/kg or a 9 nmol/kg dose of the peptide of SEQ IDNO: 18 demonstrated a significant decrease in ambient glucose by Day 4(−29% vs. vehicle; P<0.05). Mice that were given 9 nmol/kg of thepeptide of SEQ ID NO: 19 demonstrated an approximate 40% decrease inglucose levels on Day 4 (compared to vehicle control mice; P<0.05), anapproximate 39% decrease in glucose levels on Day 7 (compared to vehiclecontrol mice; P<0.05), and an approximate 26% decrease in glucose levelson Day 9 (compared to vehicle control mice; P<0.05).

Mice that were given 9 nmol/kg of the peptide of SEQ ID NO: 17demonstrated an approximate 48% decrease in glucose levels on Days 4 and7 (compared to vehicle control mice; P<0.05), an approximate 45%decrease in glucose levels on Day 9 (as compared to vehicle controlmice; P<0.05). Mice that were given 3 nmol/kg of the peptide of SEQ IDNO: 17 demonstrated an approximate 28% reduction in glucose levels onDay 7 (as compared to vehicle control mice; P<0.05) and an approximate19% decrease in glucose levels on Day 9 (as compared to vehicle controlmice; P<0.05).

Example 8

The in vivo effects of a peptide of the invention were confirmed inobese rhesus monkeys by administering daily for 21 days a vehiclecontrol, a commercially available product (Liraglutide), a peptide ofSEQ ID NO: 20, or a peptide of SEQ ID NO: 17. Liraglutide wasadministered at a dose of 20 μg/kg s.c., while each of the peptides ofSEQ ID NO: 17 and SEQ ID NO: 20 was administered at a dose of 3 μg/kgs.c. All peptides have comparable pharmacokinetic (t½ ˜10 h) and similarplasma protein binding in rhesus monkeys (>98%). Body weight and foodintake were measured.

As shown in FIG. 3A, obese monkeys that were administered the peptide ofSEQ ID NO: 17 exhibited superior weight loss (−1.2 kg vs. baseline andvehicle, P<0.05), compared to Liraglutide and to the peptide of SEQ IDNO: 20.

A significant decrease in food intake (expressed as % change vs.baseline in FIG. 3B), was observed following treatment with liraglutide(−13.1±1.9%) or with the peptide of SEQ ID NO: 17 (−25.7%±3.2%, P<0.05vs. Liraglutide). Following a one week wash-out, the animals treatedwith the peptide of SEQ ID NO: 17 regained much of the weight that waslost during the chronic treatment (Day 28). In FIGS. 3A and 3B, the dataare expressed as mean±SEM. * P<0.05 vs vehicle, ^P<0.05 peptide of SEQID NO: 17 vs. Liraglutide.

Example 9

The in vivo effects of a peptide of the invention were tested indiabetic rhesus monkeys (n=7) that were aged 16-26 years and having anaverage body weight of ˜22 kg. Diabetic rhesus monkeys (n=7) that wereaged 16-26 years with an average body weight of ˜22 kg were administereda vehicle control, Liraglutide at a dose of 10 μg/kg s.c. or a peptideof SEQ ID NO: 17 at a dose of 1 μg/kg s.c. for 10 days.

Body weight was measured during the study and it was found that no bodyweight effect was observed in these animals during the treatment period.

At the end of each treatment, fasting blood glucose (FBG) was measuredfirst followed by a Meal Tolerance Test (MTT) to assess glucosetolerance. As shown in FIG. 4, diabetic monkeys treated with the peptideof SEQ ID NO: 17 demonstrated a significantly decreased FBG (120±20 vs.158±15 mg/mL, peptide of SEQ ID NO: 17 vs. vehicle (−25%), P<0.05; FIG.4). Also, the monkeys treated with the peptide of SEQ ID NO: 17demonstrated an improved glucose tolerance during MTT as compared tovehicle control monkeys (FIG. 4). Data are expressed in FIG. 4 asmean±SEM. *P<0.05 versus vehicle.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A peptide comprising the amino acid sequence of: SEQ ID NO: 12 or a pharmaceutically acceptable salt thereof.
 2. A peptide of claim 1 consisting of the amino acid sequence of SEQ ID NO: 12, or a pharmaceutically acceptable salt thereof.
 3. A dimer or multimer comprising a peptide of claim 1, or a pharmaceutically acceptable salt thereof.
 4. A pharmaceutical composition comprising the peptide of claim 1, or a pharmaceutically acceptable salt thereof.
 5. The pharmaceutical composition of claim 4, further comprising an anti-diabetic or anti-obesity agent.
 6. The pharmaceutical composition of claim 5 wherein said anti-diabetic agent is selected from the group consisting of insulin, leptin, Peptide YY (PYY), Pancreatic Peptide (PP), fibroblast growth factor 21 (FGF21), Y2Y4 receptor agonists, tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron), repaglinide (Prandin), nateglinide (Starlix), metformin (Glucophage), phenformin, rosiglitazone (Avandia), pioglitazone (Actos), troglitazone (Rezulin), miglitol (Glyset), acarbose (Precose/Glucobay), exenatide (Byetta), pramlintide, vildagliptin, sitagliptin, glucokinase activators (GKA), glucagon receptor antagonists (GRA), and FBPase (fructose 1,6-bisphosphatase) inhibitors.
 7. The pharmaceutical composition of claim 5 wherein said anti-obesity agent is selected from the group consisting of phenethylamine type stimulants, phentermine, fenfluramine, dexfenfluramine, diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®), benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant (Acomplia®), oxyntomodulin, fluoxetine hydrochloride (Prozac), Qnexa, Excalia, Contrave, XENICAL (Orlistat), Cetilistat, or GT 389-255.
 8. A method of treating a disease or medical condition in a patient in need thereof, wherein the disease or medical condition is selected from the group consisting of: metabolic syndrome, diabetes, obesity, and liver steatosis, comprising administering to the patient the pharmaceutical composition of claim 4 in an amount effective to treat the disease or medical condition.
 9. A conjugate comprising a peptide of claim 1 conjugated to a heterologous moiety, or a pharmaceutically acceptable salt thereof, wherein the conjugate exhibits enhanced activity at the GLP-1 receptor, as compared to native glucagon, and exhibits at least 100-fold greater selectivity for the human GLP-1 receptor versus the GIP receptor.
 10. The conjugate of claim 9, wherein the heterologous moiety comprises one or more of: a peptide, a polypeptide, a nucleic acid molecule, an antibody or fragment thereof, a polymer, a quantum dot, a small molecule, a toxin, a diagnostic agent, or a pharmaceutically acceptable salt thereof.
 11. The conjugate of claim 10, comprising an extension of 1-21 amino acids C-terminal to the amino acid at position 31 of the peptide or variant peptide, or a pharmaceutically acceptable salt thereof.
 12. The conjugate of claim 11, wherein the extension is selected from the group consisting of: Gly, Glu, Cys, Gly-Gly, Gly-Glu, GPSSGAPPPS (SEQ ID NO: 9) or GGPSSGAPPPS (SEQ ID NO: 10), or a pharmaceutically acceptable salt thereof. 